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標題: | 鑑定APP與flotillin-1之間產生交互作用的重要區段並探討此交互作用對APP processing的影響 Identification of the regions mediating APP-flotillin-1 interaction and the effects of their interaction on APP processing |
作者: | Ya-Yun Lo 羅雅云 |
指導教授: | 孔繁璐 |
關鍵字: | 阿茲海默氏症,APP蛋白,Flotillin-1蛋白,lipid raft,Thr668磷酸化,C99/C83數值, Alzheimer’s disease,Amyloid precursor protein,Flotillin-1,lipid raft,Thr668 phosphorylation,C99/C83 ratio, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 阿茲海默氏症 (Alzheimer’s disease) 是老年失智症中最常見的一種,經由病患腦組織切片可以發現有兩個主要的特徵,一是細胞內的神經纖維糾結(neurofibrillary tangles),另一則是細胞外老年斑 (senile plaques) 的形成。老年斑的主要成份是A-beta,A-beta的產生是類澱粉前驅蛋白 (amyloid precursor protein, APP) 經由連續性的酵素 (beta-secretase和gamma-secretase) 水解而形成。研究上認為有許多因素可能參與了APP的代謝,進而影響了A-beta的生成,像是脂筏 (lipid rafts)。由本實驗室之前的研究發現,一脂筏相關蛋白flotillin-1會與APP的C端片段 (APP intracellular domain, AICD) 產生交互作用,另外也發現APP的Thr668位置的磷酸化可能會影響了APP和flotillin-1之間的交互作用,進而改變了APP在細胞內的分佈及影響APP的代謝。因此我們想要找出APP與flotillin-1之間產生交互作用的重要區段,以及探討Thr668位置的磷酸化是否會影響了APP與flotillin-1之間的交互作用,除此之外,也要觀察此交互作用是否會影響APP的代謝,最後還要研究lipid rafts在APP代謝中所扮演的角色。研究結果是,經由石英晶體微天秤系統 (quartz crystal microbalance, QCM) 證實了flotillin-1160-185可與APP產生交互作用。利用模擬Thr668磷酸化 (APPT668E和AICDT668E) 及非磷酸化 (APPT668V和AICDT668A) 狀態的 mutants分別與flotillin-1進行交互作用的分析,APP的Thr668被磷酸化後會增進兩者間的交互作用能力,並可能藉由與flotillin-1之間的交互作用把APP帶到lipid rafts上,造成APP被beta-secretase及gamma-secretase水解,也就是APP代謝傾向走amyloidogenic pathway,使得A-beta生成量增加 (我們以C99/C83數值的變化來觀察)。經由siRNA knockdown實驗發現,當flotillin-1被knockdown時,APP代謝會傾向走non-amyloidogenic pathway,這證實flotillin-1對APP的proecessing有一定的作用。利用truncated flotillin-1 mutants去進行competition studies可以推出flotillin-1和APP間的交互作用會影響APP processing。總而言之,我們初步的結果指出flotillin-1可能會藉由與磷酸化APP的交互作用而促進APP代謝走向amyloidogenic pathway。 Alzheimer's disease (AD) is the most common form of eldly dementia. It is characterized by two hallmarks in brain tissue slices of AD patients: one is intracellular neurofibrillary tangles (NFTs) and the other is extracellular senile plaques (SPs) formation. The main component of SPs is A-beta, which is generated by sequential proteolytic cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. Previous studies indicated that a number of factors, such as lipid rafts, may have been involved in APP processing, thereby affecting the A-beta production. Our earlier observations showed that AICD (APP intracellular domain) is able to interact with a lipid raft-associated protein, flotillin-1, and phosphorylation of APP at Thr668 probably affects the interaction between AICD and flotillin-1. It would change APP intracellular distribution and influence APP processing. Based on these studies, we want to identify regions of APP and flotillin-1 important for their interaction and to explore whether Thr668 phosphorylation can affect the interaction between APP and flotillin-1. In addition, we also want to see whether this interaction will affect the APP processing and to study the roles lipid rafts play in APP processing. Results from quartz crystal microbalance (QCM) studies indicated that APP can interact with flotillin-1160-185. Interaction studies using APPT668 mutants, APPT668E and T668A/V, which mimic constitutively phosphorylated and nonphosphorylated threonine, respectively, revealed that Thr668 phosphorylation will enhance the interaction between APP and flotillin-1, which in turn may promote APP translocation to lipid rafts, where it is subject to hydrolysis by beta-secretase and gamma-secretase to generate A-beta (as suggested by an increase in the C99/C83 ratio). According to siRNA knockdown studies, APP tends to be processed via the non-amyloidogenic pathway when flotillin-1 was knocked down. This result suggests that flotillin-1 may play a role in APP processing. Preliminary results from competition studies using truncated flotillin-1 mutants suggested APP processing may be modulated by APP-flotillin-1 interaction. In summary, our preliminary results suggest that flotillin-1 may promote amyloidogenic processing of APP through interacting with APP which is phosphorylated on Thr668. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17004 |
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顯示於系所單位: | 藥學系 |
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