The Role of γ-Secretase Activating Protein (GSAP) and Imatinib in the Regulation of γ-Secretase Activity and Amyloid-β Generation
The Role of γ-Secretase Activating Protein (GSAP) and Imatinib in the Regulation of γ-Secretase Activity and Amyloid-β Generation
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DOI：
10.1074/jbc.m112.370924
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发表时间：
2013-01-25
影响因子：
4.8
通讯作者：
Beher, Dirk
中科院分区：
文献类型：
--
作者：
Hussain, Ishrut;Fabregue, Julien;Beher, Dirk
γ-分泌酶是包含早老素、nicastrin、早老素增强子2和前咽缺陷1的大酶复合物，其介导大量蛋白质（包括淀粉样前体蛋白和Notch）的膜内蛋白水解。最近，一种新的γ-分泌酶激活蛋白（GSAP）被确定为与γ-分泌酶和淀粉样前体蛋白的C-末端片段相互作用，以选择性地增加淀粉样β蛋白的产生。在这项研究中，我们进一步表征了内源性和外源性GSAP在体外调节γ-分泌酶活性和淀粉样β蛋白产生中的作用。N2 a细胞中GSAP表达的敲低降低了淀粉样蛋白β水平。相反，在表达淀粉样蛋白前体蛋白的HEK细胞或N2 a细胞中GSAP的过表达对淀粉样蛋白-β的产生没有明显的影响。同样地，纯化的重组GSAP在两种不同的体外γ-分泌酶测定中对淀粉样蛋白-β的产生没有影响。在随后的细胞研究中，伊马替尼（一种激酶抑制剂，据报道可阻止GSAP与淀粉样前体蛋白C-末端片段的相互作用）观察到淀粉样蛋白-β水平呈浓度依赖性降低。然而，在免疫共沉淀研究中，GSAP和淀粉样前体蛋白的C-末端片段之间没有明显的相互作用。此外，亚慢性给予大鼠伊马替尼对脑淀粉样蛋白β水平没有影响。总之，这些发现表明GSAP和伊马替尼在调节γ-分泌酶活性和β-淀粉样蛋白生成中的作用是不确定的。
gamma-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel gamma-secretase activating protein (GSAP) was identified that interacts with gamma-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-beta production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of gamma-secretase activity and amyloid-beta production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-beta levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-beta generation. Likewise, purified recombinant GSAP had no effect on amyloid-beta generation in two distinct in vitro gamma-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-beta levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-beta levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of gamma-secretase activity and amyloid-beta generation are uncertain.