Glu332 in the Nicastrin ectodomain is essential for γ-secretase complex maturation but not for its activity
Glu332 in the Nicastrin ectodomain is essential for γ-secretase complex maturation but not for its activity
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DOI：
10.1074/jbc.m803040200
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发表时间：
2008-07-18
影响因子：
4.8
通讯作者：
de Strooper, Bart
中科院分区：
文献类型：
--
作者：
Chavez-Gutierrez, Lucia;Tolia, Alexandra;de Strooper, Bart
γ-分泌酶复合物负责整合膜蛋白的蛋白水解。已经提出Nicastrin作为复合物的底物受体，谷氨酸332（人中的Glu（333））作为底物的α-氨基末端基团的阴离子结合位点。推定的结合位点位于Nicastrin的氨基肽酶样结构域内。Glu（332）被认为是位于这些肽酶活性部位的外肽酶Glu的对应物。虽然Glu（332）可以结合底物的α-氨基末端基团，但我们假设，与M28-氨基肽酶类似，Nicastrin的推定结合位点中的其他残基也应该参与相互作用。令人惊讶的是，这些残基的诱变仅微弱地影响APP和Notch底物的体内加工。此外，E332 Q突变完全消除了阴离子α-氨基末端结合功能，保持完全活性。当我们引入先前表征的E332 A突变时，我们发现γ-分泌酶复合物水平强烈降低，但剩余的复合物表现出与野生型复合物一样的活性。我们在两个独立的体外试验中证实，E332 A突变体的比酶活性与野生型复合物相当。因此，Glu（332）对复合物成熟的影响至关重要，而不是对底物识别的影响。此外，其他Nicastrin突变体，旨在阻碍或改变基本上假定的结合口袋，影响只有轻微的γ-分泌酶活性。因此，这些研究表明Glu（332）的主要作用是在γ-分泌酶的成熟和组装中，而不是在底物的识别中。
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.