Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ

Abstract

The amyloid β-peptide (Aβ) of Alzheimer's disease (AD) is generated by proteolysis within the transmembrane domain (TMD) of a C-terminal fragment of the amyloid β protein-precursor (APP CTFβ) by the γ-secretase complex. This processing produces Aβ ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of Aβ peptides is the result of successive γ-secretase cleavages, with endoproteolysis first occurring at the ε sites to generate Aβ48 or Aβ49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of Aβ: the primary Aβ49-46-43-40 line and a minor Aβ48-45-42-38 line. The major secreted Aβ species are Aβ40 and Aβ42, and an increased proportion of the longer, aggregation-prone Aβ42 compared to Aβ40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of ε site endoproteolysis and carboxypeptidase trimming of CTFβ by γ-secretase. We determined that the C-terminal negative charge of the intermediate Aβ49 does not play a role in its trimming by γ-secretase. Peptidomimetic probes suggest that γ-secretase has S1', S2', and S3' pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the ε sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic ε cleavage of CTFβ. We also show that instability of the CTFβ TMD helix near the ε site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by γ-secretase. In addition, we found that CTFβ dimers are not endoproteolyzed by γ-secretase. These results support a model in which initial interaction of the array of residues along the undimerized single helical TMD of substrates dictates the site of initial ε cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.

Figure 1. γ-secretase trims Aβ49 and Aβ49 C-amide to generate primarily Aβ40

(A) The transmembrane domain of APP CTFβ is processed by γ-secretase by sequential cleavage at the ε, ζ, and γ sites to generate the Aβ peptides with the indicated C-termini. ε site cleavage at position Aβ49 leads to subsequent trimming at Aβ46, 43, and 40 to generate primarily secreted Aβ40, while ε cleavage at position Aβ48 leads to cleavage at position Aβ45, Aβ42, and Aβ38 to generate primarily secreted Aβ42. (B) A model for γ-secretase trimming of long Aβ intermediates every three residues. The negatively charged C-terminus, generated upon scission at the ε site, would move three residues due to attraction to positive charges on the cytosolic side of the enzyme, setting up the next cut at the subsequent cleavage site. This process would then repeat until the Aβ is secreted from the membrane. (C) Aβ40 and Aβ42 production from Aβ49 or Aβ49 C-amide and CHAPSO-solubilized membranes containing overexpressed γ-secretase. Aβ40 and Aβ42 products were measured by ELISA. +I reactions contain the γ-secretase inhibitor L-685,458. Mean +/− S.E.M, n=2. (D) Concentration-dependent γ-secretase inhibition by transition-state analogue inhibitors with a methyl ester or a carboxylic acid at their C-termini. Mean +/− S.E.M. n=3.

Figure 2. γ-secretase has S1’, S2’, and S3’ pockets

Concentration-dependent γ-secretase inhibition by hydroxyethylurea-type transition-state analogue γ-secretase inhibitors with and without a p3’ substituent. Mean +/− S.E.M. n=3.

Figure 3. Deletion of residues around the ε sites does not alter the primary Aβ cleavage products

(A) C100-FLAG deletion mutants. One, two, or three residues on either side of the primary ε site (between Aβ residues 49 and 50) were deleted. The transmembrane domain is in red. The predicted sites of ε and γ site cleavage are in bold and indicated with arrows. (B) A summary of the mass spectrometric analysis of the Aβ and AICD products of each deletion mutant. The C-termini of the Aβ species detected by mass spectrometry are indicated by solid vertical bars above, the N-termini of AICD species are indicated by dashed bars below; the larger bars indicate products with larger peaks in the mass spectra. (C) Aβ products of C100-FLAG deletion mutants were separated by bicine urea gel electrophoresis. Aβ signal was visualized by western blotting with anti-Aβ antibody 6E10. A representative result of five independent experiments is shown. (D) AICD production from the deletion mutants was monitored by anti-FLAG western blotting and was quantified and normalized to WT. For each mutant, AICD production was significantly lower than for WT. *p<0.05, **p<0.01, ***p<0.001 versus WT. Mean +/− S.E.M. n=2.

Figure 4. Depth within the TMD is not a determinant of ε-site specificity

The top row of helices demonstrates how the cleavage sites would be predicted to shift if initial ε site cleavage always occurred three residues within the transmembrane domain. The bottom row of helices summarizes the results obtained in Figure 3.

Figure 5. Helical instability between the ε and ζ sites is important for endoproteolysis at the ε site

(A) Helix-promoting (LL) and helix-destabilizing motifs (GG and GA) were inserted between cleavage sites. The mutated residues are indicated in red. (B) C100-FLAG substrates with GG, GA, and LL mutations between the ε and ζ sites (in red) were used as substrates in in vitro γ-secretase assays. AICD production was monitored by anti-FLAG western blotting. AICD signal was quantified by densitometry and normalized to WT. ** p<0.01, ***p<0.001 versus WT. Mean +/− S.E.M. n=4. A representative blot is shown, and the lower blot is a longer exposure of the upper blot. (C) Cleavage reactions were carried out with the indicated concentrations of substrate. For each blot, an equal amount of a standard AICD loading control (LC) was included. AICD signal was quantified by densitometry and normalized to that of the LC. Data were fit using GraphPad prism 6 non-linear regression analysis. Representative blots and curves of three independent experiments are shown.

Figure 6. Effect of helical propensity between the ε and ζ sites on trimming

(A) Aβ generated in enzyme assays using the same substrates in Figure 5 were analyzed by bicine urea gel electrophoresis. Aβ was detected by 6E10 western blot. A representative blot of four independent experiments is shown. (B) A summary of the mass spectrometric analysis of the Aβ and AICD species generated in these reactions. The data are labeled as in Figure 3B.

Figure 7. Effect of helical propensity between ζ and γ sites and γ and γ’ sites on endoproteolysis at the ε site

(A) Enzyme assays were performed using C100-FLAG substrates with helix-promoting and helix-destabilizing residues between the ζ and γ and γ and γ’ sites. AICD production was detected by anti-FLAG western blot. AICD signal was quantified by densitometry and normalized to WT. Mean +/− S.E.M. n=5. A representative blot is shown No significant differences in AICD levels were found between the mutants and WT, with the exception of the γ to γ’ site GG and GA mutants, for which AICD product levels were too low to be consistently quantified. (B) All C100-FLAG substrates were purified by anti-FLAG affinity chromatography, and the purifications were examined by SDS-PAGE. The GG γ to γ’ mutant runs as a dimer and is shown with a purification of WT C100-FLAG, which largely runs as a monomer, for comparison.

Figure 8. Effects of helical propensity between ζ and γ sites and γ and γ’ sites on trimming

(A) Aβ products generated from the same substrates used in Figure 7 were analyzed by bicine urea gel electrophoresis. Asterisks indicate Aβ products running differently than the Aβ standards due to the presence of the mutations. These bands were identified by mass spectrometry as follows: the products in the GG ζ- to-γ lane, GA ζ-to-γ lane, and LL ζ to γ lane are Aβ45 containing each respective mutation, and the products in the LL γ-to-γ’ lane are Aβ42 and Aβ43 containing the LL mutation. A representative blot of three independent experiments is shown. (B) A summary of the mass spectrometric analysis of the Aβ and AICD products generated from these substrates. The data are labeled as in Figure 3B.