Dissociation between the processivity and total activity of γ-secretase: implications for the mechanism of Alzheimer's disease-causing presenilin mutations

Abstract

The amyloid β-peptide (Aβ), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is produced from the amyloid β-protein precursor (APP) through consecutive proteolysis by β- and γ-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aβ production and increase the ratio of the aggregation-prone 42-residue form (Aβ42) to the 40-residue form (Aβ40). The connection between these two effects is not understood. Besides Aβ40 and Aβ42, γ-secretase produces a range of Aβ peptides, the result of initial cutting at the ε site to form Aβ48 or Aβ49 and subsequent trimming every three or four residues. Thus, γ-secretase displays both overall proteolytic activity (ε cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild-type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature, and salt concentration that reduced the overall activity of the wild-type enzyme did not consistently result in increased proportions of longer Aβ peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aβ peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD mutant complexes, representing a broad range of effects on overall activity, Aβ42:Aβ40 ratios, and ages of disease onset, were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ε site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aβ peptides.

Figure 1. Effects of pH on γ-secretase activity and processivity

γ-Secretase assays were performed from pH 5.5 to 9.0. In order to span this range, three buffering agents were used: MES (pH 5.5, 6.0, and 6.5), HEPES (pH 7.0, 7.5, and 8.0), and bicine (pH 8.0, 8.5, and 9.0). A) The total activity of γ-secretase was measured using anti-FLAG Western blotting to detect AICD-FLAG (inset). Bands were quantified and normalized to HEPES buffer pH 7.0. At pH 8.0 the data from HEPES buffer is displayed, although bicine at pH 8.0 gave the same result (Fig S1). Starred columns are significantly different from pH 7.0. B) The Aβ fragments produced in these assays were analyzed on bicine/urea gels (upper) and identified using known Aβ standards (Std). Quantitation of each band allowed for determination of the relative contribution of each of the Aβ species (lower). The data at pH 8.0 is from the HEPES buffered assay. The bands for pH 9.0 were too faint to be reliably quantified. C) Aβ38 and Aβ46+ show significant changes in contribution to total Aβ with changing pH. Statistical significance was determined by the unpaired two-tailed student t-test. P < 0.05. The data is the average from three independent experiments.

Figure 2. Effects of salinity on γ-secretase activity and processivity

Shown are γ-secretase assays performed with 0 to 150 mM NaCl or Na₂SO₄. A) The total activity of γ-secretase was measured using anti-FLAG Western blotting to detect AICD-FLAG (inset). Bands were quantified and normalized to 150 mM salt. Starred columns are significantly different from 150 mM salt. B) The Aβ fragments produced in these assays were analyzed on bicine/urea gels (upper). Quantitation of each band allowed for determination of the relative contribution of each of the Aβ species (lower). C) Aβ46+ shows significant changes in contribution to total Aβ. Starred column is significantly different from 150 mM NaCl. Statistical significance was determined by the unpaired two-tailed student t-test. P < 0.05. The data is the average from two independent experiments.

Figure 3. Effects of temperature on γ-secretase activity and processivity

Shown are results from γ-secretase assays performed between 20 °C and 55 °C. A) The total activity of γ-secretase was measured using anti-FLAG Western blotting to detect AICD-FLAG (inset). Bands were quantified and normalized to 35 °C. Starred columns are significantly different from 35 °C. B) The Aβ fragments produced in these assays were analyzed on bicine/urea gels (upper). Quantitation of each band allowed for determination of the relative contribution of each of the Aβ species (lower). C) Aβ40 and Aβ43 show significant changes in contribution to total Aβ. Statistical significance was determined by the unpaired two-tailed student t-test. P < 0.05. The data is the average of two independent experiments.

Figure 4. PS1 FAD mutants differ in their rate of AICD formation

A) Schematic of location and age of onset for PS1 FAD mutants used in this study. B) Solubilized membranes containing PS1 FAD mutants were normalized for levels of NTF and analyzed by Western blot for other γ-secretase components and presenilin endoproteolysis. From top to bottom: anti-GST to detect GST-nicastrin, anti-presenilin N-terminal fragment (NTF) to detect full-length presenilin and NTF, and anti-presenilin C-terminal fragment (CTF) to detect both exogenous human and endogenous hamster CTF. C) Equal amounts of wild-type and FAD mutant γ-secretase were included in C99 activity assays, and total activity was assessed using anti-FLAG Western blotting to detect AICD-FLAG (lower blot). Bands were quantified and normalized to a wild-type PS1 control (upper blot). All PS1 FAD mutants, except L286V, show significant statistical difference relative to wild type (graph, starred columns). Statistical significance was determined by the unpaired two-tailed student t-test. P < 0.05. The data is the average of three independent experiments.

Figure 5. FAD mutations result in large differences in Aβ distribution

The Aβ peptides produced by each FAD-mutant and wild type γ-secretase were separated on bicine/urea SDS-PAGE and visualized by anti-Aβ 6E10 Western blotting. * Denotes band artifacts as previously reported (ref). B) Quantitation of each band allowed for determination of the relative contribution of each of the Aβ species. C) Comparison of changes in Aβ40 and Aβ42+Aβ43 reveals PS1 FAD mutants that decrease Aβ40, increase Aβ42+Aβ43, or both. Starred columns are significantly different from wild-type. Statistical significance was determined by the unpaired two-tailed student t-test. P < 0.05. The data is the average of four independent experiments.

Figure 6. Lack of correlation between total γ-secretase activity and the general distribution of Aβs

A) The processivity index (sum of short Aβ38 and Aβ40, divided by the sum of long Aβs, Aβ42 to Aβ46+) was plotted in order of total activity for each FAD mutant. B) The processivity index and total activity was plotted for the γ-secretase assays run at different temperatures.