APP C端片段與flotillin-1間交互作用之研究

Abstract

阿茲海默氏症屬於一種神經退化疾病，有兩種重要特徵:其一是神經纖維糾結，另一則是老年斑。老年斑的主要成分為Aβ，是amyloid precursor protein (APP) 經過一連串酵素序列性切割作用後的產物。因此，了解APP的代謝過程對於預防及治療阿茲海默氏症相當重要。 我們之前以酵母雙雜交實驗找出了一些可與APP的C端 (AICD) 產生交互作用的人類腦蛋白質，其中之一是一個在腦部組織有大量表現的lipid raft marker，flotillin-1。以一株神經母細胞瘤細胞株 (SH-SY5Y) 的蛋白質萃取液進行免疫共沉澱實驗，證實了APP C端片段與flotillin-1之間確實有associated，並非direct interaction之證據。此外，我們利用一些flotillin-1的deletion mutants，試圖藉由酵母雙交實驗和免疫共沉澱實驗，找出flotillin-1上與AICD作用、以及與其他flotillins間形成聚合體相關的重要區域。實驗結果顯示，一個位於flotillin-1 N端的putative oligomerization domain (137-188 a.a.) 對其與AICD之交互作用是重要的。另一個位於flotillin-1 C端的putative oligomerization domain (189-282 a.a.) 則是對其產生聚合體是重要的。另外，有關APP的磷酸化與flotillin-1形成聚合體之間關係的實驗正在進行中。

Alzheimer’s disease (AD) is a neurodegenerative disease. Two hallmarks of AD are neurofibrillary tangles (NFTs) and senile plaques (SPs). The main component of SPs is Aβ, the product of a serial enzymatic digestion of amyloid precursor protein (APP). Therefore, it is important to understand the processing of APP to prevent or cure AD. Human brain proteins interacting with APP-intracellular-C-terminal-domain (AICD) were identified using a yeast two-hybrid screening assay. One of the protein candidates that may interact with AICD is flotillin-1, a lipid raft marker expressed predominantly in brain tissues. The association between APP and its C-terminal-fragment (CTF) with flotillin-1 was confirmed by co-immunoprecipitation experiment using the protein extracts from a neuroblastoma cell line SH-SY5Y. In addition, a series of deletion mutants of flotillin-1 were generated to identify region(s) on flotillin-1 important for the interaction between AICD and flotillin-1 or between flotillins by yeast two-hybrid assay and co-immunoprecipitation experiment. Our result suggests that a putative oligomerization domain near the N-terminus of flotillin-1 (137-188 a.a.) may be crucial for the AICD-flotillin-1 interaction. The other putative oligomerization domain near the C-terminus of flotillin-1 (189-282 a.a.) may be crucial for flotillin-1-flotillin-1 interaction. Experiments to elucidate whether phosphorylation of APP is associated with oligomerization of flotillin-1 are still ongoing.