Notch and the amyloid precursor protein are cleaved by similar γ-secretase(s)
Notch and the amyloid precursor protein are cleaved by similar γ-secretase(s)
复制标题
DOI：
10.1021/bi026888g
复制
发表时间：
2003-01-14
期刊：
影响因子：
2.9
通讯作者：
Wolfe, MS
中科院分区：
文献类型：
--
作者：
Kimberly, WT;Esler, WP;Wolfe, MS
分泌酶是一种膜内切割蛋白酶，其底物包括Notch和淀粉样前体蛋白（APP）。根据最初的遗传和药理学数据，负责切割这两种蛋白质的γ分泌酶活性似乎是相同的。然而，切割位点和序列特异性的明显差异引发了Notch和APP γ样蛋白水解相似程度的问题。为了直接解决这一问题，我们建立了一种体外γ分泌酶活性测定方法，该方法可以切割基于APP和notch的底物C100Flag和NI00Flag。特异γ -分泌酶抑制剂、显性阴性γ -分泌酶制剂和抗体共免疫沉淀物的分析都表明，这些底物具有相同的切割作用。最重要的是，我们发现这些底物阻止了彼此的切割，这表明相同的γ分泌酶复合物可以切割任何一种蛋白质。最后，我们提供的证据表明，这两种底物都在跨膜区域的两个不同区域被切割。这些数据解决了一些明显的冲突，并强烈表明Notch和APP被相同的酶水解。
gamma-Secretase is an intramembrane-cleaving protease whose substrates include Notch and the amyloid precursor protein (APP). On the basis of initial genetic and pharmacologic data, the gamma-secretase activity responsible for cleavage of both proteins appears to be identical. However, apparent differences in the cleavage site and in sequence specificity raise questions about the degree of similarity between Notch and APP gamma-like proteolysis. In an effort to resolve this issue directly, we established an in vitro gamma-secretase activity assay that cleaves both APP- and Notch-based substrates, C100Flag and NI00Flag. Analysis with specific gamma-secretase inhibitors, dominant-negative gamma-secretase preparations, and antibody co-immunoprecipitations all demonstrated identical cleavage of these substrates. Most importantly, we found that these substrates prevented cleavage of each other, indicating that the same gamma-secretase complex can cleave either protein. Finally, we provide evidence that both substrates are cut at two distinct regions in the transmembrane domain. These data resolve some of the apparent conflicts and strongly indicate that Notch and APP are proteolyzed by the same enzyme(s).