| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 94 === It is believed that the increasing production of citrate can provide lipid precursors for proliferating tumor cells especially for membrane lipid formation. In addition, recent studies have found that the citrate synthase (CS) activity is markedly overexpressed in many human cancers, such as pancreatic ductal and bladder carcinoma cells, strongly suggesting that the CS activity is associated with cell proliferation in cancer cells. Thus, CS may serve as a putative therapeutic target for cancers. Citrate synthase (CS) is one of the key regulatory enzymes in the energy-generating metabolic pathway. It catalyzes the condensation of oxaloacetate (OAA) and acetyl coenzyme A (acetyl-CoA) to form citrate in the tricarboxylic acid (TCA) cycle. The 28.6 kb of human CS gene consists of twelve exons that express two transcriptional variants, citrate synthase isoform a (CSa) and isoform b (CSb). In this study, we are interested in the expression level of different tissues between the two transcriptional variants. CSa is localized in mitochondrial matrix; however, its mitochondrial targeting sequence has not been analyzed by molecular technology. To dissect the mitochondrial targeting sequence of CSa, we have constructed a CSa-enhanced green fluorescence protein (EGFP) fusion protein expression vector and two red fluorescence protein (DsRed2) expression vectors that specifically targeted to mitochondria and peroxisomes. After co-transfection, the results confirmed that CSa is localized in mitochondria. Furthermore, we have expressed fusion constructs of truncated or site-mutated CSa and flurorescent proteins. The results indicated that the N-terminal 27 amino acid sequence in CSa is essential and sufficient for mitochondrial import. The reduction of the net positive charge in this segment decreased mitochondrial specificity and the mutants were distributed throughout the cytosol. However, when all serine and threonine in this segment were mutated, the mitochondrial specificity would not be affected. To dissect the mechanism of mitochondria import, we co-transfected the expression vector targeting to mitochondria and shRNAs against Tom complex, and found that these shRNAs have effect on the efficacy of motchondria import. In addition, to study the biological function of CS, we have used an efficient siRNA screening strategy to identify extremely potent siRNA molecules. These siRNAs could induce CS mRNA degradation and results in the reduction of CS activity. The effective siRNA may be a potential therapeutic strategy for the cancers that CS activity is significantly elevated.
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