Dev Kumlesh K, Chatterjee Sandipan, Osinde Maribel, Stauffer Daniela, Morgan Hannah, Kobialko Monika, Dengler Uwe, Rueeger Heinrich, Martoglio Bruno, Rovelli Giorgio
Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland.
Eur J Pharmacol. 2006 Jul 1;540(1-3):10-7. doi: 10.1016/j.ejphar.2006.04.011. Epub 2006 Apr 15.
The intramembrane-cleaving proteases (I-CLiPs) presenilin-1 and -2 (PS1 and PS2), signal peptide peptidase (SPP) and the Site-2 protease (S2P) catalyze critical steps in cell signaling and are implicated in diseases such as Alzheimer's disease, hepatitis C virus (HCV) infection and cholesterol homeostasis. Here we describe the development of a cellular assay based on cleavage of the transmembrane sequence of the HCV core protein precursor, releasing intra- and extra-cellular signals that represent sequential signal peptidase and SPP cleavage, respectively. We find that the SPP inhibitor (Z-LL)2-ketone (IC50 = 1.33 microM) and the gamma-secretase potent inhibitors NVP-AHW700-NX (IC50 = 51 nM) and LY411575 (IC50 = 61 nM) but not DAPT dose dependently inhibited SPP but not signal peptidase cleavage. Our data confirm that type II orientated substrates, like the HCV transmembrane sequence, are sequentially cleaved by signal peptidase then SPP. This dual assay provides a powerful tool to pharmacologically analyze sequential cleavage events of signal peptidase and SPP and their regulation.
膜内裂解蛋白酶（I-CLiPs）早老素-1和-2（PS1和PS2）、信号肽酶（SPP）以及位点2蛋白酶（S2P）催化细胞信号传导中的关键步骤，并与诸如阿尔茨海默病、丙型肝炎病毒（HCV）感染和胆固醇稳态等疾病相关。在此，我们描述了一种细胞检测方法的开发，该方法基于HCV核心蛋白前体跨膜序列的裂解，释放分别代表信号肽酶和SPP裂解的细胞内和细胞外信号。我们发现，SPP抑制剂（Z-LL）2-酮（IC50 = 1.33 microM）以及γ-分泌酶强效抑制剂NVP-AHW700-NX（IC50 = 51 nM）和LY411575（IC50 = 61 nM）呈剂量依赖性地抑制SPP，但不抑制信号肽酶的裂解，而DAPT则无此作用。我们的数据证实，像HCV跨膜序列这样的II型定向底物先被信号肽酶裂解，然后被SPP裂解。这种双重检测方法为从药理学角度分析信号肽酶和SPP的顺序裂解事件及其调控提供了一个强大的工具。
