Figure 1.
Generation of stable cell lines overexpressing all human γ-secretase components with FAD-linked PS1 variants.
MEF PS1/2−/− were stably co-transduced with lentiviral vectors carrying genes encoding hNCT-V5, Flag-hPEN2, hAPH1aL-HA and clones were isolated by limiting dilution to generate a cell line, designated as γ- PS1/2, that overexpresses high amount of the three subunits. γ- PS1/2 MEFs were further transduced with hPS1 variants harbouring FAD-linked mutations or mutations in the catalytic aspartate residue(s), or PS1-WT, and cloned. Each clone, derived form the γ- PS1/2, was conveniently named according to the mutation present in PS1 preceded by the symbol γ and followed by the number of the clone (γ-MEF) in order to distinguish them from wild-type MEF (WT MEF) and MEF PS1/2−/−. Two clones per γ-secretase variant were selected for characterization. (A–B) Whole cell protein extracts of the different cell lines were prepared in 1% NP40-HEPES buffer, separated by SDS-PAGE on 4–12% Bis-Tris or 12% Tris-Glycine gels and analysed by immunostaining to detect the γ-secretase core components NCT (NCT164), PS1 (NTF, MAB1563; CTF; MAB5232), APH1aL-HA (3F10), and Flag-PEN2 (M2) (A), and endogenous APP (A8717) (B). β-Actin was used as a loading control. Each lane represents one selected clone. CTF: C-terminal fragment, FL: full-length, im.: immature NCT; m.: mature NCT, N: N-glycosylated, NTF: N-terminal fragment, O: O-glycosylated.
Figure 2.
Aβ production in cell lines overexpressing human γ-secretase components with FAD-linked PS1 variants.
WT MEF, γ-MEF and γ - PS1/2 were transduced with an APP-based substrate corresponding to the 99 C-terminal residues of human APP fused to the APP signal peptide in N-terminus (SPA4CT [36]) and a Flag Tag in C-terminus. Cell proteins were extracted in 1% NP40-HEPES buffer, separated by SDS-PAGE on 12% Tris-Glycine gels and analysed by immunostaining with an antibody targeting the C-terminal part of APP (A8717) (A, C). Aβ1–40 and Aβ1–42 levels were also measured in the corresponding cell culture media (B, D). Data corresponds to three independent experiments (Mean ± SEM).
Figure 3.
Enzymatic activity of partially purified γ-secretase complexes with FAD-linked PS1 mutants.
(A) γ-Secretase activity assays performed with γ-MEF and γ - PS1/2 microsomal extracts prepared in 1% CHAPSO-HEPES buffer. Equal protein levels from the different extracts were diluted to 0.25% CHAPSO-HEPES buffer and incubated for 4 h at 37°C with lipids and 1 µM of recombinant human APP-based substrate (C100-Flag). Samples were analyzed by SDS-PAGE and immunostained with anti-Flag (M2) or anti-PS1 (MAB1563). The relative amounts of AICD-Flag generated in the reactions, reflecting γ-secretase activity, were estimated by densitometry. PS1 immunostaining was used to assess the amount of input material. (B) Equal amounts of microsomal proteins were immunoprecipitated overnight at 4°C with either anti-Flag M2 or anti-HA affinity resins, and submitted to a C100-His assay according to the same protocol as in (A). Protein samples were separated by SDS-PAGE and analysed by immunostaining for γ-secretase subunits ((NCT164 (NCT), MAB1563 (PS1-NTF), or UD1 (PEN2)). AICD-His cleavage products were immunostained with an anti-APP-CTF antibody (A8717). *Indicates a non-specific band corresponding to the IgG light chains. (C) Aβ1–40 and Aβ1–42 were quantified by sandwich ELISA and represented in pg/mL (left Y-axis) or in percentage (right Y-axis) of the mean of Aβ1–40 levels generated by the two wild-type clones. Aβ1–42/Aβ1–40 ratios are indicated on the top of the bars. The results were confirmed in three independent experiments and a representative dataset is shown.
Figure 4.
High-grade purification of human γ-secretase complexes with FAD-linked PS1 mutants.
(A) Schematic representation of the γ-secretase purification process. Briefly, Presenilin double-knockout MEFs were used to first generate cell lines that stably overexpress human γ-secretase complexes containing different PS1 variants. Next, these cell lines were used for a multi-step purification procedure as described in the material and methods. (B) Blue-Native PAGE analysis of purified γ-secretase complexes made of different PS1 variants. Equal volumes of the different purified γ-secretase preparations were separated by native-PAGE on a 4–16% Bis-Tris gel, and stained with silver nitrate (top panel), or immunostained for NCT (NCT164, middle panel) or PS1-NTF (ab10281, bottom panel) as indicated. γ-Secretase complexes appeared on the gel as high molecular weight complexes (HMWCs) of ∼350 kDa. Note that the levels of HMWCs were similar for all clones. (C) Equal volumes of purified γ-secretase complexes with FAD-linked PS1 mutants were separated under denaturing conditions (SDS-PAGE) and immunostained with anti-NCT (NCT164), anti-PS1-NTF (MAB1563), anti-PS1-CTF (MAB5232), anti-HA (3F10), or anti-Flag (M2) antibodies. Two independent purifications were performed on each clone with similar results. A representative dataset is shown.
Figure 5.
Enzymatic activity of highly purified γ-secretase complexes with FAD-linked or aspartate PS1 mutants.
Equal amounts of the different purified γ-secretase preparations characterized in Figure 4 were tested for activity on C100-Flag, as described in Figure 3. The resulting cleavage products were separated by SDS-PAGE and detected by immunostaining with an anti-Flag antibody (M2) for C100-Flag or AICD-Flag (A), and by sandwich ELISA for Aβ1–40 or Aβ1–42 (B). Note that the levels of Aβ produced from FAD-linked γ-secretase complexes were all in the non-linear range of the ELISA standards, close to the detection limit. Whenever possible, Aβ1–42/Aβ1–40 ratios were quantified and indicated on the top of the bars. Two independent purifications were performed on each clone and similar results were obtained. A representative dataset is shown.