TNF-α經由活化JNK MAPK pathway增強γ-secretase活性分子機制之研究

Abstract

Amyloid-β precursor protein (APP)經過β-secretase和γ-secretase相繼作用後產生Amyloid-β (Aβ)，是阿茲海默症致病機制中非常重要的一步。γ-secretase是負責在APP的transmembrane domain執行最後一步的蛋白水解反應，許多研究都認為這是阿茲海默症致病機制中最關鍵的ㄧ步。近來已有證據顯示γ-secretase為一個由presenilin (PS)、nicastrin (NCT)、Aph-1和Pen-2所組成的多蛋白複合體，且此四種蛋白的完善組合為其酵素活性的充要條件。先前的證據已指出一些cytokines能夠刺激γ-secretase的活性，其中又以tumer necrosis factor-α (TNF-α)最為有效。在最初的研究中，我們發現TNF-α透過JNK-dependent MAPK pathway活化γ-secretase，我們接著想看看cytokines對於γ-secretase的調控是否來自於JNK對於此蛋白水解酶的磷酸化所做的改變。為了提高對於γ-secretase磷酸化改變的解析度，我們建立了一細胞株大量表現有His-tag的NCT，以及正常的PS1、Aph-1、Pen-2和APP，因此可以用HIS-SelectedTM Cobalt Affinity Gel有效地純化整個γ-secretase複合體。在此，我們提出一些證據顯示TNF-α透過JNK促進PS1和NCT上的serine/threonine的磷酸化程度，同時活化γ-secretase；若用JNK的抑制劑─SP600125抑制JNK的活性，則會減低TNF-α對於PS1和NCT所引起的磷酸化。再者，活化的JNK能夠與γ-secretase一起被純化出來，並且活化的JNK可以在體外實驗中促進PS1和NCT的磷酸化。我們接著在PS1上找到一可能由JNK所調控的磷酸化位置，擁有此PS1突變的細胞表現出降低的γ-secretase活性。由我們的發現可以推論在TNF-α所引起γ-secretase的磷酸化中，JNK為其細胞中一重要的調停者，可能藉由直接與γ-secreatase相互作用來達到調控其鄰酸化程度與活性的目的。

Amyloid-β (Aβ), which is generated through β- and γ-secretase-mediated proteolysis of amyloid precursor protein (APP), plays a critical role in the pathogenesis of Alzheimer’s disease (AD). γ-Secretase which cleaves Amyloid-β precursor protein (APP) in its transmembrane domain catalyzes the final proteolytic step in the generation of Aβ. Recent evidence has demonstrated that γ-secretase is a multiprotein complex composed of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2 and that all four proteins are essential and sufficient for the proteolytic activity of γ-secretase. Previous evidence has shown that several cytokines can stimulate γ-secretase activity, and tumor necrosis factor-α (TNF-α) is the most potent one. In this study, we initially show that TNF-α activates γ-secretase via a JNK-dependent MAPK pathway. We then seek to determine whether this cytokine-elicited regulation of γ-secretase is due to alteration in the JNK-dependent phosphorylation of this protease. To improve the efficiency on visualizing the alteration in phosphorylation of γ-secretase, we have generated a CHO-derived cell line (γNCT-36) that stably co-expresses a His-tagged NCT along with PS1, Aph-1, Pen-2 and APP. The complexes of γ-secretase can then be efficiently pulled down by HIS-SelectedTM Cobalt Affinity Gel through the affinity of His-tagged NCT. Here, we present evidence that TNF-α elicits JNK-dependent serine/threonine phosphorylation of PS1 and NCT in γNCT-36 cells, concomitant with the stimulation of γ-secretase activity. Blocking JNK activity with a potent JNK inhibitor (SP600125) can reduce TNF-α-triggered phosphorylation of PS1 and NCT. Consistently, the activated JNK can be co-purified with γ-secretase complexes, and promotes the phosphorylation of PS1 and NCT in an in vitro kinase assay. A putative JNK-induced phosphorylation site of PS1 has been identified, and cells harboring this PS1 mutant exhibit decreased γ-secretase activity. Our findings suggest that JNK is an intracellular mediator of TNF-α-elicited phosphorylation of γ-secretase and may directly interact with γ-secretase to modulate its phosphorylation levels and hence its activity.