脂筏(lipid rafts)對γ-secretase分解類澱粉前驅蛋白之 功能性研究

The role of lipid rafts in the spatial and functional correlations between γ-secretase and the proteolysis of amyloid precursor protein C-terminal fragments

林帝宇(Ti-Yu Lin)

指導教授 ： 李士傑

共同指導教授 ： 廖永豐

國立臺灣大學/生命科學院/動物學研究研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02936

摘要

阿茲海默症病人腦中有顯著的類澱粉蛋白斑塊。這些斑塊是由類澱粉蛋白(Aβ, amyloid β-peptide)沉澱堆積所形成。Aβ是amyloid precursor protein (APP)經由β-及γ-secretases連續分解所生成。APP先經由β-secretase分解形成APP-99-amino acid carboxy-terminal APP fragment (C99)，而APP-C99再經由γ-secretase分解形成Aβ及APP intracellular domain (AICD)。脂筏(Lipid rafts)是細胞膜上特殊的微小構造，膽固醇、sphingolipids富含於其中而賦予其對界面活性劑的抗性。這些微小的構造藉由提供特化的環境而調控著細胞中許多的生理反應，例如:訊息傳遞、細胞膜上的分子運輸、病原體對細胞的入侵等等。Caveolae是一種屬於lipid raft的構造，可藉由其中的結構蛋白caveolin-1來維持其在細胞膜上的穩定性。為了要研究lipid rafts是否會影響γ-secretase的活性，我們發展了無細胞及活細胞的γ-secretase活性分析法。利用methyl-β-cyclodextrin (MβCD)可除去細胞膜上的膽固醇而破壞lipid rafts的特性。在無細胞的環境下分析，我們發現這會降低γ-secretase在細胞膜上對APP-C-terminal fragments (CTFs)的分解。我們因此預期，對APP-CTFs有活性的γ-secretase存在於lipid rafts中。然而，在活細胞的環境下分析，我們卻發現以MβCD破壞lipid rafts反而會刺激γ-secretase對APP-C99的分解。同時，以大量表現caveolin-1來穩定細胞中caveolae的結構則會減低γ-secretase對APP-C99的分解。為了解答在不同環境下分析所產生的矛盾，我們著手於分析在無細胞的環境下，lipid rafts能否維持其在膜上的結構。結果顯示，lipid rafts在我們進行無細胞γ-secretase活性分析法的過程中已遭到破壞。所以，有活性的γ-secretase脫離了在細胞內原有的lipid rafts而更易接近APP-CTFs。我們也研究了APP-C99在細胞內的分布狀況，只有一小部分的APP-C99會在lipid rafts中。雖然破壞了lipid rafts會降低γ-secretase對APP-CTFs的活性，但是增加的γ-secretase與APP-C99在細胞中的反應機率卻超過了活性減低的效應而使得APP-C99的分解增加。綜合實驗的結果，我們推論，在細胞中，lipid rafts不但賦予γ-secretase對APP-CTFs的活性，卻也同時隔開了兩者，使得反應不易發生。

關鍵字

阿茲海默症 ； 類澱粉蛋白 ； 膽固醇 ； 脂筏(Lipid raft) ； γ-secretase活性分析法

並列摘要

Amyloid plaque is the hallmark in Alzheimer’s disease (AD) brains. It is composed of amyloid β-peptide (Aβ) deposits. Aβ is produced through a series of cleavages of amyloid precursor protein (APP) by β- and γ-secretases. The γ-cleavage of the 99-amino acid carboxy-terminal APP fragment (APP-C99), the product from the β-cleavage of APP, produces Aβ and the APP intracellular domain (AICD). Many studies have suggested that lipid rafts are involved in the regulation of γ-secretase activity. Lipid rafts are compartmentalized membrane microdomains enriched in cholesterol and sphingolipids, and are resistant to nonionic detergent extraction at 4 °C. These microdomains allow specific protein interactions that modulate many cellular processes, including signal transduction, membrane trafficking, and pathogen entry. Caveolae are one subtype of lipid rafts which is coated by the structural protein, caveolin-1. We investigated the functional role of lipid rafts in the regulation of γ-secretase activity using both cell-free and cell-based γ-secretase assays. Disruption of lipid rafts by the cholesterol-extracting agent, methyl-β-cyclodextrin (MβCD), decreased γ-secretase-catalyzed cleavage of APP-C-terminal fragments (CTFs) in cell-free conditions. We therefore hypothesized that γ-secretase-mediated cleavage of APP-CTFs is localized in lipid rafts. However, disruption of lipid rafts stimulated γ-cleavage of APP-C99 in cell-based conditions. Consistent with the cell-based study, stabilizing caveolae by overexpressing caveolin-1 in T20 cells decreased the γ-cleavage of APP-C99. To resolve this discrepancy, we analyzed the raft-mediated compartmentalization of isolated microsomal membranes that provides the platform for the γ-secretase-catalyzed proteolysis of APP-CTFs. Our data indicated that the compartmentalization mediated by lipid rafts is disrupted during the preparation of microsomal membranes and that the active γ-secretase confined to lipid rafts is released to all APP-CTFs in isolated microsomal membranes. We also analyzed the raft-mediated partitioning of APP-C99 in intact cells. Only a small pool of APP-C99 was associated with lipid rafts. Although the disruption of lipid rafts decreased γ-secretase activity for APP-CTFs in our cell-free assays, the resulting increased access of this active protease to APP-C99 might overwhelm this effect to stimulate AICD generation and Aβ secretion we detected in our cell-based assays. Together, these results suggest that γ-secretase possesses activity for APP-CTFs in lipid rafts and is spatially segregated from APP-C99 in cultured cells.

並列關鍵字

Alzheimer’s disease ； amyloid β-peptide ； γ-secretase ； caveolin-1 ； lipid rafts

參考文獻

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