Characterization of the Reconstituted -Secretase Complex from Sf9 Cells Co-Expressing Presenilin 1, Nacastrin, aph-1a, and pen-2

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• 作者：Lili Zhang ; Julie Lee ; Lixin Song ; Xiaoyan Sun ; Jie Shen ; Giuseppe Terracina ; and Eric M. Parker
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：March 22, 2005
• 年：2005
• 卷：44
• 期：11
• 页码：4450 - 4457
• 全文大小：456K
• 年卷期：v.44,no.11(March 22, 2005)
• ISSN：1520-4995

文摘

-Secretase catalyzes the proteolytic processing of a number of integral membrane proteins,including amyloid precursor protein (APP) and Notch. The native -secretase is a heterogeneous populationof large membrane protein complexes containing presenilin 1 (PS1) or presenilin 2 (PS2), aph-1a or aph-1b, nicastrin, and pen-2. Here we report the reconstitution of a -secretase complex in Sf9 cells by co-infection with baculoviruses carrying the PS1, nicastrin, pen-2, and aph-1a genes. The reconstituted enzymeprocesses C99 and the Notch-like substrate N160 and displays the characteristic features of -secretasein terms of sensitivity to a -secretase inhibitor, upregulation of A42 production by a familial Alzheimer'sdisease (FAD) mutation in the APP gene, and downregulation of Notch processing by PS1 FAD mutations.However, the ratio of A42:A40 production by the reconstituted -secretase is significantly higher thanthat of the native enzyme from 293 cells. Unlike in mammalian cells where PS1 FAD mutations cause anincrease in A42 production, PS1 FAD missense mutations in the reconstitution system alter the cleavagesites in the C99 substrate without changing the A42:A40 ratio. In addition, PS1E9 is a loss-of-function mutation in both C99 and N160 processing. Reconstitution of -secretase provides a homogeneoussystem for studying the individual -secretase complexes and their roles in A production, Notch processingand AD pathogenesis. These studies may provide important insight into the development of a new generationof selective -secretase inhibitors with an improved side effect profile.