γ-Secretase modulators exhibit selectivity for modulation of APP cleavage but inverse γ-secretase modulators do not
γ-Secretase modulators exhibit selectivity for modulation of APP cleavage but inverse γ-secretase modulators do not
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DOI：
10.1186/s13195-020-00622-5
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发表时间：
2020-05-19
影响因子：
9
通讯作者：
Ran, Yong
中科院分区：
文献类型：
--
作者：
Lessard, Christian B.;Rodriguez, Edgardo;Ran, Yong
背景γ-分泌酶是一种多蛋白蛋白酶，可切割淀粉样蛋白前体（APP）和其他I型跨膜蛋白。它有两个催化亚基，早老素1和2（PS1和2）。在我们之前的报告中，我们观察到PS1和PS2介导的选择底物裂解的细微差异，以及PS1与PS2对选择γ-分泌酶抑制剂（GSI）在各种底物上的抑制效力略有不同。在这项研究中，我们研究了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调节γ-分泌酶的持续合成能力。我们接下来使用其中PSEN 1或PSEN 2被选择性敲除的HEK 293 T细胞系来研究持续合成能力和对GSM和iGSM的响应。方法在无细胞γ-分泌酶切割试验中，将重组底物与CHAPSO增溶的CHO或HEK 293 T细胞膜在合适的缓冲液中与GSMs或iGSMs孵育。对于基于细胞的测定，将编码底物的cDNA转染到HEK 293 T细胞中。然后用GSM或iGSM处理细胞，并收集条件培养基。通过ELISA和质谱法测量来自无细胞和基于细胞的测定的A β和A β样肽产生。结果GSMs对APP的作用具有高度的选择性，而iGSMs对多种底物的作用更为混杂。令人惊讶的是，iGSMs实际上似乎在选择的底物上起到类似GSI的作用。用PSEN 1或PSEN 2敲除HEK 293 T的数据显示，PS1比PS2具有更高的持续合成能力和对GSM的响应，但PS2对iGSM具有更高的响应。结论总的来说，这些数据表明GSM可能具有有限的靶向毒性。此外，他们还表明，iGSMs可以作为底物选择性GSI，为确定某些γ分泌酶介导的信号传导事件的底物选择性抑制剂提供了潜在的新途径。随着越来越多的人担心长期β-分泌酶抑制剂受到基于靶点的毒性的限制，这些数据支持继续开发GSM作为AD抑制剂。
Background gamma-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 gamma-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether gamma-secretase modulators (GSMs) and inverse gamma-secretase modulators (iGSMs) modulate gamma-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 gamma-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. A beta and A beta-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 gamma-secretase-mediated signaling events. With growing concerns that long-term beta-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics.