Activity-dependent isolation of the presenilin-γ-secretase complex reveals nicastrin and a γ substrate
Activity-dependent isolation of the presenilin-γ-secretase complex reveals nicastrin and a γ substrate
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DOI：
10.1073/pnas.052436599
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发表时间：
2002-03-05
影响因子：
11.1
通讯作者：
Wolfe, MS
中科院分区：
文献类型：
--
作者：
Esler, WP;Kimberly, WT;Wolfe, MS
早老素异源二聚体显然含有伽马分泌酶的活性部位，这是一种参与Notch受体和淀粉样β前体蛋白跨膜加工的多位天冬氨酸蛋白酶。尽管这种酶对胚胎发育和阿尔茨海默病的发病机制至关重要，但它的特征很难确定，主要是因为它是一种由完整的膜蛋白组成的多组分复合体。在这里，功能性的伽马分泌酶复合体是通过使用固定化的活性位点定向的蛋白酶抑制物来分离的。在与蛋白酶活性密切相关的条件下，早老素异源二聚体和尼卡斯汀与该抑制物特异结合，而其他几个与早老素相互作用的蛋白(γ-连环素、钙烯二聚体和早老素相关蛋白)不结合。此外，抗尼古丁抗体免疫共沉淀法从洗涤剂溶解的微粒体内获得了γ-分泌酶活性。出乎意料的是，C83，伽马分泌酶的主要内源性淀粉样β前体蛋白底物，也与复合体定量相关。这些结果提供了直接的生化证据，表明尼古丁是活性伽马分泌酶复合体的成员，表明β-连环蛋白、钙化蛋白和早老素相关蛋白不是伽马活性所必需的，并提示了底物-蛋白酶相互作用的一种前所未有的机制。
Presenilin heterodimers apparently contain the active site of gamma-secretase, a polytopic aspartyl protease involved in the transmembrane processing of both the Notch receptor and the amyloid-beta precursor protein. Although critical to embryonic development and the pathogenesis of Alzheimer's disease, this protease is difficult to characterize, primarily because it is a multicomponent complex of integral membrane proteins. Here the functional gamma-secretase complex was isolated by using an immobilized active site-directed inhibitor of the protease. Presenilin heterodimers and nicastrin bound specifically to this inhibitor under conditions tightly correlating with protease activity, whereas several other presenilin-interacting proteins gamma-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated gamma-secretase activity from detergentsolubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-beta precursor protein substrate of gamma-secretase, was also quantitatively associated with the complex. These results provide direct biochemical evidence that nicastrin is a member of the active gamma-secretase complex, indicate that beta-catenin, calsenilin, and presenilin-associated protein are not required for gamma activity, and suggest an unprecedented mechanism of substrate-protease interaction.