Citron M, Teplow D B, Selkoe D J
Department of Neurology, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Neuron. 1995 Mar;14(3):661-70. doi: 10.1016/0896-6273(95)90323-2.
Cerebral deposition of amyloid beta protein (A beta) is an early and critical feature of Alzheimer's disease. Here we analyze the substrate requirements of proteases ("beta-secretases") that cleave the beta-amyloid precursor protein (beta APP) at the N-terminus of A beta (Asp-597 of beta APP695) in intact human cells. The cleavage requires a membrane-bound substrate but tolerates shifts in the distance of the hydrolyzed bond from the membrane. The major protease has a minimum recognition region of Val-594 to Ala-598; most substitutions in this sequence strongly decrease or eliminate A beta production. Only the Swedish familial Alzheimer's disease mutation (K595N/M596L) strongly increases A beta production. Moreover, in this mutant but not in the wild type, the entire cytoplasmic tail with its reinternalization signals can be deleted without affecting A beta N-terminal cleavage, consistent with the concept that cleavage of this mutant occurs in a different cellular compartment than that of wild-type molecules. Our results have important implications for current intensive approaches to develop assays for and identify enzymes with beta-secretase activity.
淀粉样β蛋白（Aβ）在大脑中的沉积是阿尔茨海默病早期的关键特征。在此，我们分析了完整人类细胞中蛋白酶（“β-分泌酶”）的底物需求，这些蛋白酶在Aβ（βAPP695的Asp-597）的N端切割β-淀粉样前体蛋白（βAPP）。这种切割需要膜结合底物，但能容忍水解键与膜之间距离的变化。主要蛋白酶的最小识别区域为Val-594至Ala-598；该序列中的大多数取代会强烈降低或消除Aβ的产生。只有瑞典家族性阿尔茨海默病突变（K595N/M596L）会强烈增加Aβ的产生。此外，在该突变体中而非野生型中，带有内化信号的整个细胞质尾巴可以被删除而不影响Aβ的N端切割，这与该突变体的切割发生在与野生型分子不同的细胞区室这一概念一致。我们的结果对于当前开发β-分泌酶活性检测方法和鉴定具有β-分泌酶活性的酶的密集研究方法具有重要意义。
