Processing of Notch and amyloid precursor protein by γ-secretase is spatially distinct
Processing of Notch and amyloid precursor protein by γ-secretase is spatially distinct
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DOI：
10.1073/pnas.0408007101
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发表时间：
2004-12-07
影响因子：
11.1
通讯作者：
Li, YM
中科院分区：
文献类型：
--
作者：
Tarassishin, L;Yin, YI;Li, YM
γ-分泌酶活性与含早老素（PS）的大分子复合物有关。PS是否含有γ-分泌酶活性位点一直存在争议。一个挑战是找到参与细胞表面活性γ-分泌酶复合物的PS，其中一些底物似乎被加工。在这项研究中，我们开发了一个完整的细胞光标记技术，允许直接可视化的活性γ-分泌酶在细胞表面。我们证明了活性γ-分泌酶存在于质膜中。此外，PS1异源二聚体在细胞表面被仅与活性γ-分泌酶结合的有效抑制剂特异性地光标记。我们还利用小分子探针探索了淀粉样前体蛋白（APP）和Notch的γ-分泌酶的细胞加工位点。MRL 631是一种不能穿透细胞膜的γ-分泌酶抑制剂，可显著阻断γ-分泌酶介导的Notch切割，但对APP加工几乎没有影响。这些结果表明，Notch在细胞表面加工，而大多数APP由细胞内γ-分泌酶加工。此外，抑制剂首先靶向质膜中的γ-分泌酶进行Notch加工，而不是APP，这一事实将对治疗阿尔茨海默病和癌症的药物开发具有重要意义。
gamma-Secretase activity is associated with a presenilin (PS)-containing macromolecular complex. Whether PS contains the active site of gamma-secretase has been controversial. One challenge is to find PS that is engaged in the active gamma-secretase complex at the cell surface, where some substrates appear to be processed. In this study, we developed an intact cell photolabeling technique that allows the direct visualization of active gamma-secretase at the cell surface. We demonstrated that active gamma-secretase is present in the plasma membrane. Moreover, the PS1 heterodimer is specifically photo-labeled at the cell surface by a potent inhibitor that binds to only the active gamma-secretase. We also explored the cellular processing sites of gamma-secretase for amyloid precursor protein (APP) and Notch by using small molecular probes. MRL631, a gamma-secretase inhibitor that is unable to penetrate the cell membrane, significantly blocks gamma-secretase-mediated Notch cleavage but has little effect on APP processing. These results indicate that Notch is processed at the cell surface and that the majority of APP is processed by intracellular,gamma-secretase. Furthermore, the fact that inhibitors first target gamma-secretase in the plasma membrane for Notch processing, and not for APP, will have important implications for drug development to treat Alzheimer's disease and cancer.