Mentrup Torben, Häsler Robert, Fluhrer Regina, Saftig Paul, Schröder Bernd
Biochemical Institute, Christian Albrechts University of Kiel, Otto-Hahn-Platz 9, D-24118, Kiel, Germany.
Institute of Clinical Molecular Biology, Christian Albrechts University of Kiel, Schittenhelmstr. 12, D-24105, Kiel, Germany.
Traffic. 2015 Aug;16(8):871-92. doi: 10.1111/tra.12287. Epub 2015 May 6.
During regulated intramembrane proteolysis (RIP) a membrane-spanning substrate protein is cleaved by an ectodomain sheddase and an intramembrane cleaving protease. A cytoplasmic intracellular domain (ICD) is liberated, which can migrate to the nucleus thereby influencing transcriptional regulation. Signal peptide peptidase-like (SPPL) 2a and 2b have been implicated in RIP of type II transmembrane proteins. Even though SPPL2a might represent a potential pharmacological target for treatment of B-cell-mediated autoimmunity, no specific and potent inhibitors for this enzyme are currently available. We report here on the first quantitative cell-based assay for measurement of SPPL2a/b activity. Demonstrating the failure of standard Gal4/VP16 reporter assays for SPPL2a/b analysis, we have devised a novel system employing β-galactosidase (βGal) complementation. This is based on detecting nuclear translocation of the proteolytically released substrate ICDs, which results in specific restoration of βGal activity. Utilizing this potentially high-throughput compatible new setup, we demonstrate nuclear translocation of the ICDs from integral membrane protein 2B (ITM2B), tumor necrosis factor (TNF) and CD74 and identify secreted frizzled-related protein 2 (SFRP2) as potential transcriptional downstream target of the CD74 ICD. We show that the presented assay is easily adaptable to other intramembrane proteases and therefore represents a valuable tool for the functional analysis and development of new inhibitors of this class of enzymes.
在调节性膜内蛋白水解（RIP）过程中，一种跨膜底物蛋白被胞外域裂解酶和膜内裂解蛋白酶切割。一个细胞质内结构域（ICD）被释放出来，它可以迁移到细胞核从而影响转录调控。信号肽酶样（SPPL）2a和2b与II型跨膜蛋白的RIP有关。尽管SPPL2a可能是治疗B细胞介导的自身免疫的潜在药理学靶点，但目前尚无针对该酶的特异性强效抑制剂。我们在此报告首个基于细胞的定量测定SPPL2a/b活性的方法。由于标准的Gal4/VP16报告基因测定法无法用于SPPL2a/b分析，我们设计了一种采用β-半乳糖苷酶（βGal）互补的新系统。这基于检测经蛋白水解释放的底物ICD的核转位，其导致βGal活性的特异性恢复。利用这种可能与高通量兼容的新设置，我们证明了来自整合膜蛋白2B（ITM2B）、肿瘤坏死因子（TNF）和CD74的ICD的核转位，并确定分泌型卷曲相关蛋白2（SFRP2）为CD74 ICD潜在的转录下游靶点。我们表明所提出的测定法很容易适用于其他膜内蛋白酶，因此是用于此类酶的功能分析和新型抑制剂开发的有价值工具。
