| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 98 === Apolipoprotein A-I (apoAI), expressed and secreted by intestine and liver, is the major protein component of high-density lipoprotein (HDL) which participates in transportation of phospholipid and cholesterol from peripheral cells back to liver (a process called reverse cholesterol transport). Low circulating apoAI levels were considered to be an independent risk factor for cardiovascular disease and are closely associated with low HDL cholesterol levels. Previous proteomic studies in our lab have identified novel post-translational phosphorylations of apoAI at Ser31, Ser142, Thr161, Thr197, and Ser228 sites. The specific aim of this study was to elucidate the effects of phosphorylation modification on apoAI functions in terms of protein secretion. Human prepro-apoAI (267 amino acids) fused with a C-terminal FLAG-tag. Ala substitutions were conducted to the five candidate sites and expression in different cell lines, including CHO-K1, HEK 293T, and Huh7. The expression and secretion of the mutated AI-FLAG were different in these cells. The results suggested that expression and secretion of apoAI are influenced by cell-type specific factors. Ala or Glu substitutions were further introduced to the shRNA-resistant AI-FLAG mutant construct which could avoid targeting by shAI-3 and shAI-4. Expression of these mutated AI-FLAG in endogenous apoAI knockdown HepG2 cells showed that phosphorylation on Ser31 and Ser228 residues may play a major role in regulation of apoAI secretion. The phophorylated apoAI could be immunoprecipitated with antibodies recognizing phosphorylated serine, indicating that apoAI was phosphorylated at Ser residues. In an experiment with co-transfection of apoAI and kinases showed that overexpression of GSK3β decreased the secretion level of apoAI, indicated that apoAI may be the target of this kinase or GSK3β might regulate apoAI secretion via an indirect mechanism. In conclusion, this study has identified two phosphorylation sites (Ser31, and Ser228) which might regulate the secretion ability of apoAI and GSK3β might participate in the control of apoAI secretion.
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