Palmitoylated APP Forms Dimers, Cleaved by BACE1.
Palmitoylated APP Forms Dimers, Cleaved by BACE1.
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DOI：
10.1371/journal.pone.0166400
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Kovacs DM
中科院分区：
文献类型：
--
作者：
Bhattacharyya R;Fenn RH;Barren C;Tanzi RE;Kovacs DM
阿尔茨海默病大脑中Aβ生成和沉积的主要限速步骤是bace1介导的淀粉样前体蛋白（APP）的裂解（β-裂解）。我们之前报道过APP在e1 -外域的两个半胱氨酸残基（Cys186和Cys187）上发生棕榈酰化。在体外和体内，总APP的8-10%被棕榈酰化。棕榈酰化APP （palAPP）在抗洗涤剂脂筏中比总APP更倾向于β-裂解。已知蛋白质棕榈酰化可促进蛋白质二聚化。由于APP在其e1 -外结构域的二聚化会导致bace1介导的APP分裂升高，我们现在研究了APP的棕榈酰化是否会影响其二聚化，以及这是否会导致蛋白β-分裂升高。在这里，我们报告了超过90%的palAPP是二聚体，而只有约20%的APP形成二聚体。palapp二聚体主要是顺式的，而总APP二聚体是顺式和反式的。PalAPP形成二聚体的效率是总APP的4.5倍。过表达棕榈酰化酶DHHC7和DHHC21增加PalAPP水平和Aβ释放，也增加了APP在细胞中的二聚体。相反，在共免疫沉淀和FLIM/FRET实验中，药理学抑制剂抑制APP棕榈酰化可降低APP二聚化。最后，体外bace1活性测定表明，与总APP相比，棕榈酰化依赖的APP二聚化促进了富脂耐洗剂细胞膜（DRMs）中APP的β-裂解。最重要的是，sAPPβ-sAPPβ二聚体的生成依赖于APP-棕榈酰化，而总sAPPβ的生成不依赖于此。由于BACE1对palAPP二聚体的偏好高于总APP， palAPP二聚体可能作为APP β-裂解抑制剂的新靶点，而不是BACE1抑制剂。
A major rate-limiting step for Aβ generation and deposition in Alzheimer’s disease brains is BACE1-mediated cleavage (β-cleavage) of the amyloid precursor protein (APP). We previously reported that APP undergoes palmitoylation at two cysteine residues (Cys186 and Cys187) in the E1-ectodomain. 8–10% of total APP is palmitoylated in vitro and in vivo. Palmitoylated APP (palAPP) shows greater preference for β-cleavage than total APP in detergent resistant lipid rafts. Protein palmitoylation is known to promote protein dimerization. Since dimerization of APP at its E1-ectodomain results in elevated BACE1-mediated cleavage of APP, we have now investigated whether palmitoylation of APP affects its dimerization and whether this leads to elevated β-cleavage of the protein. Here we report that over 90% of palAPP is dimerized while only ~20% of total APP forms dimers. PalAPP-dimers are predominantly cis-oriented while total APP dimerizes in both cis- and trans-orientation. PalAPP forms dimers 4.5-times more efficiently than total APP. Overexpression of the palmitoylating enzymes DHHC7 and DHHC21 that increase palAPP levels and Aβ release, also increased APP dimerization in cells. Conversely, inhibition of APP palmitoylation by pharmacological inhibitors reduced APP-dimerization in coimmunoprecipitation and FLIM/FRET assays. Finally, in vitro BACE1-activity assays demonstrate that palmitoylation-dependent dimerization of APP promotes β-cleavage of APP in lipid-rich detergent resistant cell membranes (DRMs), when compared to total APP. Most importantly, generation of sAPPβ-sAPPβ dimers is dependent on APP-palmitoylation while total sAPPβ generation is not. Since BACE1 shows preference for palAPP dimers over total APP, palAPP dimers may serve as novel targets for effective β-cleavage inhibitors of APP as opposed to BACE1 inhibitors.
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