Tomasselli Alfredo G, Qahwash Isam, Emmons Thomas L, Lu Yifeng, Leone Joseph W, Lull June M, Fok Kam F, Bannow Carol A, Smith Clark W, Bienkowski Michael J, Heinrikson Robert L, Yan Riqiang
Department of Protein Sciences, Pharmacia Corporation, 301 Henrietta Street, Kalamazoo, MI 49007, USA.
J Neurochem. 2003 Mar;84(5):1006-17. doi: 10.1046/j.1471-4159.2003.01597.x.
The involvement of beta-secretase (BACE1; beta-site APP-cleaving enzyme) in producing the beta-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2' sequence, ...VKM--DA..., was replaced by ...ISY--EV... (-- = beta site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL--DAEFR. The expanded peptide, GLTNIKTEEISEISY--EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these beta-site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the beta-site. Insertion of the optimized ISY--EV motif at either the beta-site (Asp1) or beta'-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.
β-分泌酶（BACE1；β-位点淀粉样前体蛋白裂解酶）参与在阿尔茨海默病患者大脑中发现的斑块的β-淀粉样蛋白成分的产生，这推动了对这种蛋白酶进行特性描述的重大研究工作。在此，我们描述了为理解BACE1的底物特异性所开展的工作，这项工作始于对天然淀粉样前体蛋白（APP）底物及其瑞典突变体APPSw的研究，随后又纳入了氧化胰岛素B链和泛素底物。基于这些发现以及对其他合成肽的研究，我们确定，源自APP的一个十肽，其中P3 - P2'序列……VKM--DA……被……ISY--EV……（-- = 裂解β位点）取代，产生了一种底物，其被BACE1裂解的速度比相应的APPSw肽SEVNL--DAEFR快7倍。扩展肽GLTNIKTEEISEISY--EVEFRWKK被裂解的速度比其十肽对应物（加粗部分）又快7倍，并且提供了一种底物，可用于在皮摩尔浓度下检测BACE1。制备了反映这些β位点氨基酸变化 的几种APP突变体作为细胞分析的基础。APPISYEV突变体被证明是一种优于APPSw的细胞底物。基于APPISYEV的分析对于测量细胞中的BACE1活性具有高度特异性；其同源物BACE2在β位点几乎不裂解APPISYEV。在β位点（Asp1）或β'位点（Glu11）插入优化的ISY--EV基序可指导APP在这两个可及位点的细胞加工速率。因此，我们已经鉴定出了最佳的BACE1底物，这些底物将有助于阐明BACE1的细胞酶促作用，并用于设计可能对阿尔茨海默病具有治疗益处的抑制剂。
