Abstract
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.
Highlights
Protease complex cleaves the COOH-terminal 99-amino acid fragment of the amyloid precursor protein (APP)3 within the membrane at different positions, to release the COOH-terminal intracellular domain (AICD) and COOH-terminal heterogeneous amyloid- peptides (A) [4, 5]
When we introduced the previously characterized E332A mutation, we found strongly decreased ␥-secretase complex levels, but the remaining complex appeared as active as the wild-type complex
A peptides are the major component of amyloid deposits in the brains of patients with Alzheimer disease, and the longer A42 peptide generation appears to be crucial in the Alzheimer amyloid cascade [6, 7]
Summary
Antibodies—Polyclonal antibodies against mouse Ps1 NTF (B19.3), APH1a (B80.3), PEN-2 (B126.1), and APP COOH terminus (B63.3) and monoclonal 9C3 against the COOH terminus of Nicastrin have been described [26, 27]. Cells were treated overnight with 10 M GM6001 inhibitor (Sigma) to avoid C83-3xFLAG generation. MEF microsomal fractions (10 mg/ml) were solubilized in 1% CHAPSO buffer (50 mM PIPES, pH 7.0, 0.25 M sucrose, 1 mM EGTA, 1ϫ Complete protease inhibitor mixture (Roche)) and incubated on ice for 1 h. In vitro reactions with 0.8 M APP C99-3xFLAG (in 20 l final volume) were carried out in 50 mM PIPES, pH 7.0, 0.25 M sucrose, 1 mM EGTA, 1ϫ EDTA-free Complete proteinase inhibitors (Roche), 2.5% DMSO, 0.1% phosphatidylcholine, and 0.0125% phosphatidylethanolamine (N[N-(3,5-difluorophenacetyl-L-alanyl](s)-phenylglycine t-butyl ester at 37 °C. In Vitro Activity Assays Using Microsomal Preparations— MEF cell lines were transiently transfected with an expression vector for C99-3xFLAG using Lipofectamine Plus reagent (Invitrogen) and collected 48 h post-transfection. Cultures were treated overnight with 5 M inhibitor IX
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