Identification Of Tricarboxylic Acid Cyclic Enzyme Complex And Its Mechanism In Primary Dilated Cardiomyopathy

The tricarboxylic acid cycle (TCA cycle). a main energy production reaction, exists in the mitochondria of most living organisms. so the mitochondrion is called "energy factory". The classic TCA cycle is a series of chemical reactions catalyzed by the 8 enzymes, including citrate synthase. aconitase, isocitrate dehydrogenase,2-oxoglutarate dehydrogenase complex, succinyl-coA synthetase. succinate dehydrogenase, fumarase, malate dehydrogenase. In the previous study, TCA cycle was recognised as a series of reaction, but the relationship of these enzymes were ignored. With the advance in science, the relationship of TCA cycle enzymes were gradually explained, but almost studies focuced on the efficency of microbial fermentation via enhance the enzymes activitiy. In our study, we identied the protein complexes of TCA cycle by immunoprecipitation-mass spectrometry (IP-MS) in mammlian. The protein complexes of 8 enzymes are first identied in this study, and ACO2-IDH2-MDH2-CS is the core complex. This big complex also include electron transfer chain (NDUFS1. ATP5B), fatty acid oxidation (ACAA2), amino acid metabolism (GOT2) and glycolysis enzymes (GAPDH). Meanwhile, we first discover the tissue specificity of TCA cycle and the difference of TCA cycle complexes in dilated cardiomyopathy.Part I:Preparation of FH and SDHA antibodiesTo identify the protein complexes of TCA cycle, we expressed and purified FH, SDHA antigens through gene cloning, plasmid recombination, prokaryotic expression, protein purification in vitro and prepared FH and SDHA antibodies. The pMAL-c5x/FH, pMAL-c5x/SDHA plasmids were construted in vitro vector, and transformed into DE3 competent to express and purify the antigens of FH and SDHA which were used to immunize mice and rabbits. The purification antibodies were detected by Western blotting, immunofluorescence and immunoprecipitation. FH and SDHA antigens were successfully expressed and purified and antiserum was prepared and tested to reveal high specificity in immunofluorescence, Western blot and immunoprecipitation. FH antibody can recognize a variety of species, such as human, rat, mouse, pig.Part II:Reserch on TCA enzyme complexesThe novel FH and SDHA antibodies were used to explore the TCA cycle enzyme complexes including AC02, IDH2, MDH2, CS, DLST, FH, SDHA, protein-protein interaction each other, and which interacts with the enzymes involing in electron transfer chain (NDUFS1, ATP5B, GOT2), amino acid metabolism (GOT2), fatty acid oxidation (ACAAA2) and glycolysis (GAPDH). The protein-protein interactions between TCA cycle enzymes with electron transfer chain, fatty acid oxidation, amino acid metabolism and glycolysis enzymes were detected by IP-MS method, and identified by Western blot and ion-exchange liquid chromatographic column experiment that was used to explore the molecular size of TCA cycle enzyme complexes. And MS data was used to construct the interaction network of these enzymes. TCA cycle enzymes interact with each other, and assemble to the large protein complexes, which contain the core protein complexes of ACO2-IDH2-MDH2-CS. TCA cycle enzyme complexes were isolated from rat mitochondria with ion-exchange liquid chromatographic column. The interaction between TCA cycle enzymes with with NDUFS1, ATP5B, ACAA2, and GAPDH was identified by IP-MS and Western blot.Part Ⅲ:Research on tissue specificity and effects on dilated cardiomyopathy of TCA cycle enzyme complexesTCA cycle enzyme complexes have different functions in different tissues or organs. The structure of TCA cycle enzyme complexes in dilated cardiomyopathy (DCM) patients’heart is significantly different from normal heart. And the intermediate level of TCA cycle has significant difference. Comparsion of FH interactome in the rat heart tissues were performed by IP-MS method with rat liver tissues. Tissue specific expression of TCA cycle enzymes was detected by western blot between fat and heart. And we explored the differences of TCA cycle enzyme complexes in normal and DCM heart by IP-MS method, and the metabolites levels of them by HPLC-MS method. The interactome of FH protein in rat liver and heart is significantly different, and contain some urea cycle enzymes in the liver, but TCA cycle enzymes in rat heart. Structures of TCA cycle enzyme complexes in patients’heart with dilated cardiomyopathy are more loosely, not tight enough, so the complexes may be disrupted, especially the core complex. The levels of TCA cycle intermediates, such as fumaric acid, malic acid were significantly decreased.Part Ⅳ:Effects of Coreopsis tinctoria Nutt. key component-Marein on TCA cycle enzymes in insulin resistance cellsCoreopsis tinctoria Nutt., a kind of medicinal plant distributed in Hubei Province, Guangdong Province, Shaanxi Province, Xinjiang and other places of China, has functions in lower blood pressure, blood glucose and blood fat. This study explores effects of the key component of Coreopsis tinctoria Nutt.-Marein on insulin resistance induced by high glucose, which affects the function of TCA cycle enzymes and the balance of TCA cycle intermediates. Insulin resistance cell model was successfully constructed by high glucose. After treatment with marein, mRNA and protein expression of TCA cycle enzymes were detected and metabolomics change were explored with HPLC-MS method. Insulin resistance cells exhibits the decreased glucose uptake rate, glycogen content and hexokinase activity, the increased protein expression levels of PEPCK, G6Pase, TCA cycle enzymes, and imbalance in metabolic levels. But marein inhibited metabolism disorder via decreasing the protein overexpression of gluconeogenic and TCA cycle enzymes.