Abstract
gamma-Secretase is a proteolytic membrane complex that processes a variety of substrates including the amyloid precursor protein and the Notch receptor. Earlier we showed that one of the components of this complex, nicastrin (NCT), functions as a receptor for gamma-secretase substrates. A recent report challenged this, arguing instead that the Glu-333 residue of NCT predicted to participate in substrate recognition only participates in gamma-secretase complex maturation and not in activity per se. Here, we present evidence that Glu-333 directly participates in gamma-secretase activity. By normalizing to the active pool of gamma-secretase with two separate methods, we establish that gamma-secretase complexes containing NCT-E333A are indeed deficient in intrinsic activity. We also demonstrate that the NCT-E333A mutant is deficient in its binding to substrates. Moreover, we find that the cleavage of substrates by gamma-secretase activity requires a free N-terminal amine but no minimal length of the extracellular N-terminal stub. Taken together, these studies provide further evidence supporting the role of NCT in substrate recognition. Finally, because gamma-secretase cleaves itself during its maturation and because NCT-E333A also shows defects in gamma-secretase complex maturation, we present a model whereby Glu-333 can serve a dual role via similar mechanisms in the recruitment of both Type 1 membrane proteins for activity and the presenilin intracellular loop during complex maturation.
Highlights
PS-NTF or -CTF alone is an adequate measure of active ␥-secretase complexes, Chavez-Gutierrez et al [17] measured specific activity by normalizing ␥-secretase products to the sum of PS1-CTF and PEN-2 presumably due to the levels of PS-NTF/CTF by themselves being at the detection limit of Western blotting with electrochemiluminescence (ECL)
Such an approach has caveats, as normalizing to the sum of PS1 and PEN-2 does not represent a measurement of the intrinsic activity per single, active enzyme; rather, this mode of normalization instead skews the data to minimize the effects of the mutations, especially when compounded with the unreliability of ECL measurement at the detection limit of Western blotting
We sought to examine the effects of Glu-333 of NCT in the context of either a normally formed ␥-secretase complex (Fig. 1, A–D) or a “pre-matured” ␥-secretase complex that bypasses PS endoproteolysis, the last step of ␥-secretase maturation and activation (Fig. 1, E–H)
Summary
Materials—C100FLAG was custom synthesized by Upstate Biotechnology. Restriction enzymes were from New England Biolabs, Inc. The lysed cell suspension was centrifuged at 20,000 ϫ g for 20 min at 4 °C, and the resulting supernatant was incubated at 4 °C overnight with Ni-NTA beads (Qiagen) that had been prewashed with phosphate-buffered saline and 5 mM imidazole. Cells were harvested by centrifugation at 2500 ϫ g for 10 min at 4 °C, and cells were resuspended in lysis buffer H (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mg/ml lysozyme, plus Roche EDTA-free protein inhibitor mixture). The lysate was spun at 100,000 ϫ g for 40 min at 4 °C, and the resulting supernatant was diluted 10-fold in phosphatebuffered saline plus 1% Nonidet P-40 before purification by anti-FLAG antibody affinity gel chromatography (Sigma) according to manufacturer’s directions. Comparisons between wild-type and mutant NCT was performed by two-way analysis of variance
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