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 proportion of the aggregation-prone 42-residue form (Aβ42) over 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 3–4 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 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-to-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.

Evidence that Enzyme Processivity Mediates Differential Aβ Production by PS1 and PS2

“…In addition, several distinct PS1 and PS2 familial mutations influence the relative production of AICD and Aβ [10, 38, 39], supporting the notion that the production of AICD and Aβ are dissociable. Taken together, these findings suggest that processivity differences between PS1 and PS2 could account for their differential Aβ production, consistent with the finding with PS1 FAD mutations [39]. …”

Alzheimer Presenilin-1 Mutations Dramatically Reduce Trimming of Long Amyloid β-Peptides (Aβ) by γ-Secretase to Increase 42-to-40-Residue Aβ

“…We performed these assays in CHAPSO detergent (27,30) as well as with purified enzyme complexes reconstituted into lipid vesicles (28) to ensure that the results are not artifacts arising from detergent solubilization and to determine if the membrane is critical to the trimming process. We and others have previously described the use of urea polyacrylamide gel electrophoresis followed by Western blotting with an N-terminally directed anti-A␤ antibody to detect the range of C-terminal variants of A␤ produced in in vitro ␥-secretase reactions (7,14,17,31). However, trimmed A␤ generated from the synthetic long A␤ substrates could not be detected by this method because the signal from the excess substrate streaked throughout the lane, completely obscuring any signals from the A␤ products (data not shown).…”

“…Moreover, APP⑀49 was processed by ␤-or ␣-secretase and then ␥-secretase to generate quantifiable amounts of secreted A␤ or p3, an N-terminally truncated A␤ generated through ␣/␥-secretase cleavage. Therefore, we generated constructs for the expression of APP⑀49 and the previously unreported APP⑀48 and introduced them into CHO cells overexpressing all four ␥-secretase components (14). Expression of these truncated APPs was confirmed by Western blot of the cell lysates (Fig.…”

“…For the inactive mutant enzyme controls, both solubilized membranes and purified complexes were prepared from CHO D1 cells (overexpressing all four ␥-secretase components but with D257A mutant PS1 containing an N-terminal Myc tag) in the same way as above, except the first affinity purification step involved antiMyc antibody-agarose beads (Sigma). For the analysis of FAD PS1 mutant complexes, membranes were prepared from CHO cells overexpressing Pen2, Aph1, nicastrin, and either WT or mutant PS1 as previously reported (14). For these preparations, membranes were isolated from 20 confluent 15-cm plates and resuspended in 160 l of 1% CHAPSO.…”

“…1A) (12), with cleavage here releasing the APP intracellular domain (AICD). Reduction of ⑀ cleavage is seen with many, although not all, FAD presenilin mutations (7,13,14).…”