PILOT PROJECT-- IDENTIFICATION OF BETA AMYLOID PROTEIN SECRETASE

随着该实验室和其他实验室最近的证明（i）， 淀粉样β蛋白前体（β APP）由以下组成性加工： β APP分泌酶产生可识别的衍生物，（ii） 内体-溶酶体系统加工可识别的羧基端 β APP衍生物可能是途径中的重要中间体 产生淀粉样蛋白，和（iii）4 kD淀粉样β蛋白（Abeta） 通常由培养细胞产生和释放，现在是现实的， 进行关键蛋白酶的鉴定， 生产的Abeta。 在这里，我们提出的实验重点是 β-APP分泌酶的鉴定，探讨其可行性 利用反义cDNA表达快速鉴定这些蛋白酶 图书馆. 我们计划在这些实验中关注betaAPP分泌酶 因为很难分离出这种不寻常的 非常重要的酶，通常使用基于以下的体外测定： 序列特异性，并且由于β APP分泌酶的敲除， 反义cDNA几乎肯定会增加表面的水平， β APP达到可以被荧光激活细胞检测到的程度 分类

With the recent demonstration by this laboratory and others (i) that the amyloid beta protein precursor (betaAPP) is constitutively processed by betaAPP secretase to produce identifiable derivatives, (ii) that the endosomal-lysosomal system processes identifiable carboxyl-terminal betaAPP derivatives likely to be important intermediates in the pathway that produces amyloid, and (iii) that 4 kD amyloid beta protein (Abeta) is normally produced and released by cultured cells, it is now realistic to undertake the identification of the key proteases that determine the amount of Abeta produced. Here, we propose experimentation focused on the identification of betaAPP secretase that will explore the feasibility of rapidly identifying these proteases using antisense cDNA expression libraries. We plan to focus on betaAPP secretase in these experiments because it is likely to be difficult to isolate this unusual and critically important enzyme conventionally using in vitro assays based on sequence specificity and because the knockout of betaAPP secretase with antisense cDNAs will almost certainly increase the level of surface betaAPP to a degree that can be detected by fluorescence activated cell sorting.