BACKGROUND γ-Secretase is a multiprotein protease that cleaves amyloid protein precursor (APP) and other type I transmembrane proteins. It has two catalytic subunits, presenilins 1 and 2 (PS1 and 2). In our previous report, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates and slightly different potencies of PS1 versus PS2 inhibition for select γ-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether γ-secretase modulators (GSMs) and inverse γ-secretase modulators (iGSMs) modulate γ-secretase processivity using multiple different substrates. We next used HEK 293T cell lines in which PSEN1 or PSEN2 was selectively knocked out to investigate processivity and response to GSMs and iGSMs. METHODS For cell-free γ-secretase cleavage assay, recombinant substrates were incubated with CHAPSO-solubilized CHO or HEK 293T cell membrane with GSMs or iGSMs in suitable buffer. For cell-based assay, cDNA encoding substrates were transfected into HEK 293T cells. Cells were then treated with GSMs or iGSMs, and conditioned media were collected. Aβ and Aβ-like peptide production from cell-free and cell-based assay were measured by ELISA and mass spectrometry. RESULT These studies demonstrated that GSMs are highly selective for effects on APP, whereas iGSMs have a more promiscuous effect on many substrates. Surprisingly, iGSMs actually appear to act as like GSIs on select substrates. The data with PSEN1 or PSEN2 knocked out HEK 293T reveal that PS1 has higher processivity and response to GSMs than PS2, but PS2 has higher response to iGSM. CONCLUSION Collectively, these data indicate that GSMs are likely to have limited target-based toxicity. In addition, they show that iGSMs may act as substrate-selective GSIs providing a potential new route to identify leads for substrate-selective inhibitors of certain γ-secretase-mediated signaling events. With growing concerns that long-term β-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics.

中文翻译：

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γ-分泌酶调节剂表现出对APP切割的调节的选择性，但是反向γ-分泌酶调节剂没有。

背景技术γ-分泌酶是一种多蛋白蛋白酶，可裂解淀粉样蛋白前体（APP）和其他I型跨膜蛋白。它具有两个催化亚基，早老蛋白1和2（PS1和2）。在我们以前的报告中，我们观察到PS1和PS2介导的选择底物裂解的细微差异，以及在各种底物上选择的γ-分泌酶抑制剂（GSI）对PS1和PS2的抑制能力略有不同。在这项研究中，我们调查了γ分泌酶调节剂（GSM）和反向γ分泌酶调节剂（iGSM）是否使用多种不同的底物来调节γ分泌酶的合成能力。接下来，我们使用选择性敲除PSEN1或PSEN2的HEK 293T细胞系来研究生产力和对GSM和iGSM的反应。方法为了进行无细胞的γ-分泌酶裂解试验，将重组底物与CHAPSO增溶的CHO或HEK 293T细胞膜以及GSM或iGSM在合适的缓冲液中孵育。对于基于细胞的测定，将编码cDNA的底物转染到HEK 293T细胞中。然后将细胞用GSM或iGSM进行处理，并收集条件培养基。通过ELISA和质谱法测量无细胞和基于细胞的测定中的Aβ和Aβ样肽的产生。结果这些研究表明，GSM对APP的影响具有高度的选择性，而iGSM在许多基质上的混杂作用更大。出乎意料的是，iGSM实际上看起来像在选定基板上的GSI一样。剔除HEK 293T的PSEN1或PSEN2的数据表明，PS1的生产力和对GSM的响应比PS2高，但PS2对iGSM的响应更高。结论集体而言，这些数据表明，GSM很可能具有有限的基于靶标的毒性。此外，他们表明，iGSM可能充当底物选择性GSI，为识别某些γ-分泌酶介导的信号转导事件的底物选择性抑制剂提供了潜在的新途径。人们越来越担心长期的β-分泌酶抑制剂会受到基于靶标的毒性的限制，这些数据支持了GSM作为AD预防剂的持续发展。