Xia W, Zhang J, Perez R, Koo E H, Selkoe D J
Department of Neurology, Harvard Medical School, Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):8208-13. doi: 10.1073/pnas.94.15.8208.
Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes increase the production of the highly amyloidogenic 42-residue form of amyloid beta-protein (Abeta42) in a variety of cell lines and transgenic mice. To elucidate the molecular mechanism of this effect, wild-type (wt) or mutant PS1 and PS2 genes were stably transfected into Chinese hamster ovary cells expressing endogenous or transfected beta-amyloid precursor protein (APP). By immunoprecipitation/Western blot analysis, APP was consistently found to coimmunoprecipitate with PS1 or PS2 proteins. Several distinct PS1, PS2, or APP antibodies precipitated PS-APP complexes that were detectable by blotting with either APP or PS antibodies. Importantly, complex formation could be detected at endogenous protein levels in nontransfected cells. In various Chinese hamster ovary cell lines, the amounts of APP coprecipitated by PS antibodies were proportional to the expression levels of both APP and PS. APP-PS complexes also were recovered from human 293 and HS683 cells. Full maturation of APP was not required for the interaction; most APP molecules complexed with PS were solely N-glycosylated. Treatment of cells with brefeldin A or incubation at 20 degrees C did not block complex formation, suggesting that the association between APP and PS occurs in part in the endoplasmic reticulum. Complex formation was detected for both wt and mutant PS and APP proteins. Deletion of the APP C-terminal domain did not abrogate complex formation, suggesting that the interaction does not occur in the cytoplasmic domains of the proteins. Our results demonstrate that wt and mutant PS1 and PS2 proteins form complexes with APP in living cells, strongly supporting the hypothesis that mutant PS interacts with APP in a way that enhances the intramembranous proteolysis of the latter by a gamma-secretase cleaving at Abeta42.
早老素1（PS1）和早老素2（PS2）基因的突变会在多种细胞系和转基因小鼠中增加淀粉样β蛋白（Aβ42）的42个残基的高淀粉样生成形式的产生。为了阐明这种效应的分子机制，将野生型（wt）或突变型PS1和PS2基因稳定转染到表达内源性或转染的β淀粉样前体蛋白（APP）的中国仓鼠卵巢细胞中。通过免疫沉淀/蛋白质印迹分析，始终发现APP与PS1或PS2蛋白共免疫沉淀。几种不同的PS1、PS2或APP抗体沉淀出的PS-APP复合物可用APP或PS抗体印迹检测到。重要的是，在未转染细胞的内源性蛋白质水平上可以检测到复合物的形成。在各种中国仓鼠卵巢细胞系中，PS抗体共沉淀的APP量与APP和PS的表达水平成正比。APP-PS复合物也从人293和HS683细胞中回收。相互作用不需要APP完全成熟；与PS复合的大多数APP分子仅进行了N-糖基化。用布雷菲德菌素A处理细胞或在20℃孵育不会阻止复合物的形成，这表明APP与PS之间的结合部分发生在内质网中。野生型和突变型PS以及APP蛋白都检测到了复合物的形成。APP C末端结构域的缺失并没有消除复合物的形成，这表明这种相互作用不是发生在蛋白质的细胞质结构域中。我们的结果表明，野生型和突变型PS1和PS2蛋白在活细胞中与APP形成复合物，有力地支持了以下假设：突变型PS与APP相互作用，通过在Aβ42处切割的γ-分泌酶增强后者的膜内蛋白水解作用。
