Chávez-Gutiérrez Lucía, Tolia Alexandra, Maes Elke, Li Tong, Wong Philip C, de Strooper Bart
Center for Human Genetics, KULeuven, and Department for Molecular and Developmental Genetics, VIB4, Herestraat 49, Leuven, Belgium.
J Biol Chem. 2008 Jul 18;283(29):20096-105. doi: 10.1074/jbc.M803040200. Epub 2008 May 23.
The gamma-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin has been proposed to operate as the substrate receptor of the complex with the glutamate 332 (Glu(333) in human) serving as the anionic binding site for the alpha-amino-terminal group of substrates. The putative binding site is located within the aminopeptidase-like domain of Nicastrin. The Glu(332) is proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu(332) could bind the alpha-amino-terminal group of substrates, we hypothesized, in analogy with M28-aminopeptidases, that other residues in the putative binding site of Nicastrin should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of APP and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolishes the anionic alpha-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased gamma-secretase complex levels, but the remaining complex appeared as active as the wild-type complex. We confirmed in two independent in vitro assays that the specific enzymatic activity of the E332A mutant was comparable with that of the wild-type complex. Thus, Glu(332) crucially affects complex maturation rather than substrate recognition. Moreover other Nicastrin mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally gamma-secretase activity. Consequently, these studies indicate that the main role of the Glu(332) is in the maturation and assembly of gamma-secretase rather than in the recognition of the substrates.
γ-分泌酶复合物负责整合膜蛋白的蛋白水解。有人提出，尼卡斯特林作为该复合物的底物受体，谷氨酸332（人类中为Glu(333)）作为底物α-氨基末端基团的阴离子结合位点。推测的结合位点位于尼卡斯特林的氨肽酶样结构域内。有人提出Glu(332)发挥着与这些肽酶活性位点中的外肽酶Glu相对应的作用。尽管Glu(332)可以结合底物的α-氨基末端基团，但我们通过与M28-氨肽酶类比推测，尼卡斯特林推测结合位点中的其他残基也应参与相互作用。令人惊讶的是，这些残基的诱变对APP和Notch底物的体内加工仅产生微弱影响。此外，完全消除阴离子α-氨基末端结合功能的E332Q突变仍具有完全活性。当我们引入先前表征的E332A突变时，我们发现γ-分泌酶复合物水平大幅下降，但剩余的复合物似乎与野生型复合物一样具有活性。我们在两项独立的体外试验中证实，E332A突变体的特异性酶活性与野生型复合物相当。因此，Glu(332)对复合物成熟起关键作用，而非底物识别。此外，设计用于阻碍或大幅改变推测结合口袋的其他尼卡斯特林突变体，对γ-分泌酶活性仅产生轻微影响。因此，这些研究表明，Glu(332)的主要作用在于γ-分泌酶的成熟和组装，而非底物识别。
