在SH-SY5Y細胞膽固醇代謝中APP蛋白所扮演的角色

Abstract

阿茲海默症 (Alzheimer’s disease, AD)是一種神經退化性疾病，神經細胞外的Aβ堆積被視為是AD的病徵之一。近年來有許多研究指出，膽固醇在β-amyloid precursor protein (APP)蛋白水解產生Aβ的途徑中扮演重要角色。在我們先前的研究中發現APP上的CRAC序列可能與cholesterol產生交互作用。 Sterol-regulated element-binding proteins (SREBPs)是一可調控膽固醇合成的轉錄因子。當細胞中的SREBP cleavage-activating protein (SCAP)偵測到膽固醇含量降低時，SCAP會將SREBP由ER帶到Golgi，進行兩次酵素切割後釋放SREBP的N端片段(mSREBP)到細胞質中，此段SREBP可進入細胞核與位於基因調控段之sterol response elements (SREs)結合，使合成膽固醇所需的酵素之表現量增高(包含3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR))。 先前有研究指出，APP透過與SREBP產生交互作用而參與膽固醇的調控。在本研究中，利用在SH-SY5Y細胞中穩定表現與膽固醇具有不同結合力的APP，藉由觀察細胞間SREBP的表現量、SREBP在細胞內的分布情形以及HMGCR的mRNA表現量，來判斷APP與膽固醇的結合力是否影響APP與SREBP的交互作用以及mSREBP調節轉錄之功能。結果顯示，在表現與膽固醇結合力較低的APPY463I與APPS622L的SH-SY5Y細胞中，total SREBP 與 HMGCR mRNA表現量皆較表現wild-type APP之細胞為低，且SREBP maturation的比例較高，推測膽固醇可提升APP與SREBP在Golgi的結合，進而影響SREBP的processing。

Alzheimer’s disease (AD) is a neurodegenerative disorder. The extracellular aggregation of β-amyloid (Aβ) is considered one of the histopathological features of AD. Recently, numerous studies indicate that cholesterol may play an important role in APP processing. Results from our previous studies also suggest that APP could have interaction with cholesterol. Sterol-regulated element-binding proteins (SREBPs) are transcription factors capable of regulating the biosynthesis of cholesterol. When SREBP cleavage-activating protein (SCAP) senses a decrease in the cholesterol, it transfers SREBP from the ER to the Golgi where the SREBP is subject to two sequential proteolytic cleavages and releases its N-terminal fragment into the cytosol. This activated fragment could be translocated into nucleus, where it binds to the sterol response elements (SREs) in the regulatory region of the gene, thus upregulating the expression of several enzymes involved in cholesterol biosynthesis, including 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Previous study indicates that APP could regulate cholesterol biosynthesis by interacting with SREBP. The aim of this study is to determine whether APP-cholesterol interaction is important for the association of APP with SREBP and the proteolytic processing of SREBP. Therefore, we stably express APP with different binding ability to cholesterol in SH-SY5Y cell lines and exam the amount of SREBP expressed, the subcellular distribution of SREBP and the expression of HMGCR at mRNA level. Our results show that in cells overexpressing APPY463I and APPS622L, both possess a lower binding ability to cholesterol compared to the wild-type, the expression of SREBP and HMGCR mRNA are significantly lower than the wild-type. This observation suggests that cholesterol increases the APP-SREBP interaction in the Golgi and effects SREBP processing.