Department of Therapeutic Area Neurodegenerative Diseases, Merck Serono S.A., Chemin des Mines 9, 1202 Geneva, Switzerland.
J Biol Chem. 2013 Jan 25;288(4):2521-31. doi: 10.1074/jbc.M112.370924. Epub 2012 Dec 3.
γ-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 γ-secretase activating protein (GSAP) was identified that interacts with γ-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-β production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of γ-secretase activity and amyloid-β production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-β levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-β generation. Likewise, purified recombinant GSAP had no effect on amyloid-β generation in two distinct in vitro γ-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-β 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-β levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of γ-secretase activity and amyloid-β generation are uncertain.
γ-分泌酶是一种大型酶复合物，由早老素、尼卡斯特林、早老素增强子 2 和前咽缺陷 1 组成，介导包括淀粉样前体蛋白和 Notch 在内的大量蛋白质的跨膜蛋白水解。最近，一种新型 γ-分泌酶激活蛋白 (GSAP) 被鉴定出来，它与 γ-分泌酶和淀粉样前体蛋白的 C 端片段相互作用，选择性增加淀粉样β的产生。在这项研究中，我们进一步研究了内源性和外源性 GSAP 在体外调节 γ-分泌酶活性和淀粉样β产生中的作用。在 N2a 细胞中敲低 GSAP 表达会降低淀粉样β水平。相比之下，在表达淀粉样前体蛋白的 HEK 细胞或 N2a 细胞中过表达 GSAP 对淀粉样β的生成没有明显影响。同样，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β的生成没有影响。在随后的细胞研究中，用伊马替尼进行处理，伊马替尼是一种激酶抑制剂，据报道可阻止 GSAP 与淀粉样前体蛋白的 C 端片段相互作用，结果观察到淀粉样β水平呈浓度依赖性下降。然而，在共免疫沉淀研究中未发现 GSAP 与淀粉样前体蛋白的 C 端片段之间存在相互作用。此外，亚慢性给予伊马替尼给大鼠对脑内淀粉样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-分泌酶活性和淀粉样β生成中的作用尚不确定。
