Three-amino acid spacing of presenilin endoproteolysis suggests a general stepwise cleavage of gamma-secretase-mediated intramembrane proteolysis

Abstract

Presenilin (PS1 or PS2) is the catalytic component of the gamma-secretase complex, which mediates the final proteolytic processing step leading to the Alzheimer's disease (AD)-characterizing amyloid beta-peptide. PS is cleaved during complex assembly into its characteristic N- and C-terminal fragments. Both fragments are integral components of physiologically active gamma-secretase and harbor the two critical aspartyl residues of the active site domain. While the minimal subunit composition of gamma-secretase has been defined and numerous substrates were identified, the cellular mechanism of the endoproteolytic cleavage of PS is still unclear. We addressed this pivotal question by investigating whether familial AD (FAD)-associated PS1 mutations affect the precision of PS endoproteolysis in a manner similar to the way that such mutations shift the intramembrane cleavage of gamma-secretase substrates. We demonstrate that all FAD mutations investigated still allow endoproteolysis to occur. However, the precision of PS1 endoproteolysis is affected by PS1 mutations. Comparing the cleavage products generated by a variety of PS1 mutants revealed that specifically cleavages at positions 293 and 296 of PS1 are selectively affected. Systematic mutagenesis around the cleavage sites revealed a stepwise three amino acid spaced cleavage mechanism of PS endoproteolysis reminiscent to the epsilon-, zeta-, and gamma-cleavages described for typical gamma-secretase substrates, such as the beta-amyloid precursor protein. Our findings therefore suggest that intramembranous cleavage by gamma-secretase and related intramembrane-cleaving proteases may generally occur via stepwise endoproteolysis.

Figure 1.

PS1 FTEV is proteolytically active. A, Schematic representation of PS1 FTEV depicting the sites of FLAG epitope and TEV cleavage site insertion (green box) C-terminal to the PS cleavage site domain (red) within the large cytoplasmic loop. Anti-Flag IP allows the isolation of PS1 FTEV CTFs derived from PS endoproteolysis. To allow the identification of the respective cleavage sites, the corresponding peptides are released by subsequent TEV protease cleavage (occurring between Q and G) and subjected to MS analysis. B, Expression and endoproteolysis of PS1 FTEV were analyzed by immunoblotting of cell lysates of HEK293/sw cells stably expressing wt PS1 or PS1 FTEV using antibodies PS1-N to the PS1 N terminus (top) or antibody 3027 to the PS1 C terminus (middle panel) of PS1. Note that the endogenous PS1 CTF is replaced by the larger CTF derived from ectopically expressed PS1 FTEV. γ-Secretase activity of PS1 FTEV was analyzed by determining total Aβ levels by immunoblot analysis using antibodies 2D8 (bottom). C, γ-Secretase cleavage specificity of PS1 FTEV was assessed by IP/MS analysis of Aβ peptides from conditioned media. D, E, Expression, endoproteolysis, and γ-secretase activity of PS1 FTEV were analyzed by immunoblotting of cell lysates of PS^−/− cells transiently cotransfected with APPsw-6myc and wt PS1 or PS1 FTEV using N- and C-terminal antibodies to PS1 as in B (upper two panels), by immunoblot analysis of AICD using anti-myc antibody 9E10, and by combined immunoprecipitation/immunoblotting of Aβ using antibodies 3552/2D8 (lower two panels) (D). Profiles of Aβ peptides generated by PS^−/− cells transiently cotransfected with APPsw-6myc and wt PS1 or PS1 FTEV were analyzed from conditioned media by IP/MS analysis (E) as in C.

Figure 2.

PS endoproteolysis occurs via multiple cleavages. A, Mass spectrum of peptides derived from PS endoproteolysis of PS1 FTEV stably expressed in HEK293 cells. PS1 FTEV CTFs were isolated by anti-Flag IP and subjected to TEV protease cleavage followed by MALDI-TOF MS. Peaks are labeled with colored letters corresponding to their N-terminal amino acids. Asterisks indicate presumably N-terminally acetylated peptides. Note that no peptides were observed for the PS1 FTEV D385A active site mutant. B, Table of calculated and observed molecular masses of peptides derived from PS endoproteolysis of PS1 FTEV. C, Illustration of PS endoproteolysis sites identified. The hydrophobic region harboring the sites of PS endoproteolysis is shown in red. Arrowheads indicating the sites of PS endoproteolysis are colored according to the respective peptide peaks observed in the mass spectrum shown in A.

Figure 3.

PS1 FAD mutants affect PS endoproteolysis. A, HEK293 cells stably expressing the indicated PS1 variants were analyzed for expression and PS endoproteolysis by immunoblotting of cell lysates as in Figure 1B. Note that wt and FAD mutant PS1 FTEV are endoproteolysed into larger CTFs that replace the endogenous PS1 CTF, demonstrating that the PS1 FTEV variants undergo functional γ-secretase complex formation. B, Determination of the cleavage sites of PS1 FTEV in the presence or absence of FAD-associated mutations as described in Figure 2A. The intensity of the highest peak corresponding to endoproteolytic cleavage after position 298 (A299) was set to 100%. Parts of the mass spectra are magnified (insets) to allow a better visualization of the V293 and V296 peaks. Note that the aggressive PS1 mutations P117L, L166P, M233V, Y256S, and G384A affect PS1 endoproteolysis at common sites (amino acid 293 or 296). C, H4 cells stably expressing wt or L166P mutant PS1 FTEV were analyzed for PS endoproteolysis cleavage sites as described in B. Note that in H4 cells, cleavage at V296 and L295 is enhanced upon expression of the PS1 L166P mutant. D, PS^−/− MEF cells transiently expressing wt or L166P mutant PS1 FTEV were analyzed for PS endoproteolysis cleavage sites as described in B. E, Summary of alterations in PS endoproteolysis caused by mutant PS1 variants. Changes in cleavage site usage of FAD mutants are denoted with red (V293, V296) arrowheads. Changes in cleavage site usage at positions V293 and V296 [increased (↑), decreased (↓), or unchanged (–)] are summarized in the table.

Figure 4.

Stepwise endoproteolysis of presenilin. A, Cell lysates from HEK293 cells stably expressing the indicated constructs were analyzed for PS expression and endoproteolysis by immunoblotting as in Figure 1B. Arrows indicate MW shifts of the PS1 FTEV CTF observed for the L295D, V296D, M298D, and A299D mutants. B, Mass spectra of peptides derived from PS endoproteolysis of PS1 FTEV variants stably expressed in HEK293 cells as described in Figure 2A. The intensities of the highest peaks detected in the spectra were set to 100%. Note that the high sensitivity of the MS analysis allowed the detection of an extremely weak A299 peak also for the M292D mutant (highlighted with the box), indicating very little residual PS autoproteolysis (note difference in scale).

Figure 5.

Double aspartate mutations arrest endoproteolysis at the initial cleavage site. A, Cell lysates from HEK293 cells stably expressing the indicated PS1 FTEV constructs were analyzed for PS expression and endoproteolysis by immunoblotting as in Figure 1B. B, Mass spectra of peptides derived from PS endoproteolysis of PS1 FTEV variants stably expressed in HEK293 cells as described in Figure 2A. The intensities of the highest peaks observed in the spectra corresponding to endoproteolytic cleavage after position 291 (M292) were set to 100%. Note that double aspartate mutants arrest endoproteolytic cleavage at residue 292. Arrows indicate reduced migration of PS1 CTFs derived from L295D/A299D and V296D/A299D.

Figure 6.

PS1 mutants that support PS endoproteolysis but not γ-secretase cleavage of APP affect the catalytic domain. A, Cell lysates from HEK293 cells stably expressing the indicated PS1 constructs were analyzed for PS expression and endoproteolysis by immunoblotting as in Figure 1B. B, Mass spectra of peptides derived from PS endoproteolysis of PS1 FTEV variants stably expressed in HEK293 cells as described in Figure 2A. The intensities of the highest peaks observed in the respective spectra were set to 100%. Note that glycine mutants arrest endoproteolytic cleavage at residue 292 or 295/296. C, PS endoproteolysis of the indicated mutant PS1 constructs containing the exon 9 deletion and/or the G384P mutation and APP processing were analyzed as in Figure 1D. D, Conversion of the TNFα ICD is slowed by the SPPL2b G420A mutation and severely reduced by the SPPL2b G420P mutation. Membranes of HEK 293 cells expressing wt SPPL2b and the indicated SPPL2b variants and TNFα were incubated for the indicated time points at 37°C and formation of the intracellular domain (ICD) was monitored by immunoblotting using the anti FLAG M2 antibody. Note that improvement of the in vitro assays allowed the additional detection of ICD4 (Fluhrer et al., 2008).

Figure 7.

A model for the stepwise cleavage and activation of PS. A, Sequential endoproteolysis of the cleavage site domain encoded by exon 9. In analogy to the sequential γ-secretase cleavage of APP, PS cleavage occurs by autoproteolysis in a stepwise manner starting with an initial ε-like cleavage at amino acid 292/3, followed by a ζ-like cleavage at amino acid 295/6 and terminating with a γ-like cleavage after amino acid 298. B, Exemplification of four different classes of mutants with different impact on PS endoproteolysis and γ-secretase activity on APP substrate processing (for details, see Discussion). Yellow dots indicate the locations of the respective mutations, and the yellow Δ indicates the exon 9 deletion. C, The hydrophobic cleavage site domain may serve as a plug blocking the active site of γ-secretase, which is in a closed conformation at this state. Autoproteolytic cleavage liberates the active site allowing PS to adapt an open conformation capable to accept γ-secretase substrates for intramembrane cleavage by lateral diffusion. In B and C, only TMDs 6 and 7 of PS containing the active site domain of γ-secretase are shown for simplicity.