An AICD-based functional screen to identify APP metabolism regulators.
An AICD-based functional screen to identify APP metabolism regulators.
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DOI：
10.1186/1750-1326-2-15
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发表时间：
2007-08-24
影响因子：
15.1
通讯作者：
Saunders AJ
中科院分区：
文献类型：
--
作者：
Zhang C;Khandelwal PJ;Chakraborty R;Cuellar TL;Sarangi S;Patel SA;Cosentino CP;O'Connor M;Lee JC;Tanzi RE;Saunders AJ
阿尔茨海默病（AD）的中心事件是β-淀粉样前体蛋白（APP）的调节性膜内蛋白水解，以产生β-淀粉样（Aβ）肽和APP胞内结构域（AICD）。Aβ是淀粉样斑块的主要成分，AICD显示转录激活特性。我们已经利用AICD反式激活特性开发了一种遗传筛选，以确定APP代谢的调节因子。该筛选依赖于APP-Gal 4融合蛋白，其在正常蛋白水解时产生AICD-Gal 4。AICD-Gal 4的产生诱导Gal 4-UAS驱动的荧光素酶表达。因此，当APP代谢的调节剂被调节时，荧光素酶表达被改变。为了验证这种实验方法，我们调节α-、β-和γ-分泌酶水平和活性。通过蛋白质印迹分析测量的AICD-Gal 4水平的变化与观察到的AICD-Gal 4介导的荧光素酶活性的变化强烈且显著相关。为了确定是否可以使用AICD-Gal 4介导的荧光素酶测定来检测APP运输/成熟和早老素1内蛋白水解的已知调节剂，我们敲低了泛素1并观察到荧光素酶活性降低。我们通过Western印迹分析证实了泛素1调节AICD-Gal 4水平，并且还观察到泛素1调节总APP水平、成熟APP与未成熟APP的比率以及PS1内蛋白水解。总之，我们已经表明，该筛选可以识别已知的APP代谢调节剂，控制蛋白水解，细胞内运输，成熟和APP及其蛋白水解产物的水平。我们第一次证明了Ubiquilin 1在人神经母细胞瘤细胞系SH-SY 5 Y中调节APP代谢。
A central event in Alzheimer's disease (AD) is the regulated intramembraneous proteolysis of the β-amyloid precursor protein (APP), to generate the β-amyloid (Aβ) peptide and the APP intracellular domain (AICD). Aβ is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered. To validate this experimental approach we modulated α-, β-, and γ-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis. Taken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y.
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影响因子：
2.9
作者：
Creaven, M;Hans, F;Khochbin, S
通讯作者：
Khochbin, S
影响因子：
4.8
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Cao, XW;Südhof, TC
通讯作者：
Südhof, TC
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56.9
作者：
Cao, XW;Südhof, TC
通讯作者：
Südhof, TC
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4.8
作者：
Hass, MR;Yankner, BA
通讯作者：
Yankner, BA
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3.5
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Blacker, D;Bertram, L;Tanzi, RE
通讯作者：
Tanzi, RE