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標題: | 探討FKBP12與AICD間交互作用對APP代謝之影響 Investigation of the Effect of FKBP12-AICD Interaction on APP Processing |
作者: | Chiao-Chen Lin 林巧澄 |
指導教授: | 孔繁璐(Fan-Lu Kung) |
關鍵字: | 阿茲海默症,類澱粉前驅蛋白, FKBP12,AICD,APP processing, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | AICD,又名類澱粉前驅蛋白胞內區塊 (APP intracelluar domain),是由類澱粉前驅蛋白(amyloid precursor protein,APP) 經過α-或是β-secretase,再經由γ-secretase切割後釋放到細胞質中的片段。過去的研究發現一些chaperone會與AICD產生交互作用,Pin1即是其中一個例子。Pin1為一具有PPIase活性的chaperone蛋白,曾經有研究指出Pin1可催化APP上的pT668P669間胜肽鍵構形轉換,使其構形趨向從cis form轉為trans form,進而造成Aβ減少。之前利用酵母菌雙雜交的實驗發現免疫親合素FKBP12與AICD有交互作用,FKBP12同樣也是具有PPIase活性的chaperone,當在HEK293T細胞中過度表現FKBP12時,C99與C83的比例會改變。FKBP12結構之研究中,曾指出Asp37、Phe46、Trp59和Tyr82位於FKBP12之基質結合位置。在本研究中我們利用一系列在這些位置點突變的FKBP12突變株 (FKBP12D37V、 FKBP12F46L、FKBP12W59A、FKBP12W59L、FKBP12Y82F),來觀察究竟是FKBP12與AICD間的交互作用還是FKBP12的PPIase活性,對前述APP水解途徑的改變有較大的影響。
在此我們利用bio-layer interferometry技術來觀察不同的FKBP12 與AICD間的交互作用,並用chymotrypsin coupled-assay來測試各個FKBP12的PPIase活性。實驗結果顯示,FKBP12W59A突變株改變了野生種FKBP12原本催化的淨反應方向,同時也觀察到在COS7細胞中過度表現FKBP12W59A,會使C99/C83比值較表現野生種FKBP12時為低。依據這些結果,我們認為FKBP12的PPIase活性在其對APP的水解過程之調控中扮演著較重要的角色。FKBP12可能催化APP上T668P669之間胜肽鍵的構形從trans轉為cis,進而導致Aβ增加。此結果使我們對於AICD的生理功能和FKBP12在阿茲海默氏症中所扮演的角色有了更進一步的了解。 APP intracellular domain (AICD) is the cytoplasmic fragment released from amyloid precursor protein (APP) processed by α- or β, and γ-secretases. Previous studies indicated that some chaperones, such as Pin1, can interact with AICD. Pin1 is a chaperone with peptidyl-prolyl cis-trans isomerase (PPIase) activity. It was reported Pin1 can catalyze cis to trans isomerization of prolyl peptide bond in APP pT668P669, resulting in a decrease of Aβ. FKBP12, another chaperone and an immunophilin with PPIase activity, was found to interact with AICD by yeast two-hybrid screening. It has been shown that overexpression of FKBP12 altered C99/C83 ratio in HEK293T cells. Previous structure studies of FKBP12 indicate that the residues Asp37, Phe46, Trp59, Tyr82 are present in the substrate binding cavity of FKBP12. Here we generate a series of point mutations at those sites (FKBP12D37V, FKBP12F46L, FKBP12W59A, FKBP12W59L, FKBP12Y82F) to investigate whether the interaction between FKBP12 and AICD, or the PPIase activity is more important in altering APP processing. In this study, bio-layer interferometry technology was used to analyze the binding affinity of FKBP12 (wild-type and mutants) to AICD, and the chymotrypsin coupled-assay was used to characterize the PPIase activity of various FKBP12. Our results indicate that FKBP12W59A, the mutant which changes the overall direction of the isomerization reaction catalyzed by the wild-type FKBP12, decreases C99/C83 ratio in COS7 cell line. Based on these observations, we suggest that the PPIase activity of FKBP12 plays a more important role in the regulation of APP processing. FKBP12 may catalyze trans to cis isomerization of the prolyl peptide bond in APP T668P669, resulting in an increase of Aβ level. This result allows us to have a clearer picture of the physiological and biological function of AICD and the role FKBP12 plays in the development of Alzheimer’s disease. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56812 |
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顯示於系所單位: | 藥學系 |
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