Mutation analysis of the presenilin 1 N-terminal domain reveals a broad spectrum of gamma-secretase activity toward amyloid precursor protein and other substrates

Abstract

The γ-secretase protein complex executes the intramembrane proteolysis of amyloid precursor protein (APP), which releases Alzheimer disease β-amyloid peptide. In addition to APP, γ-secretase also cleaves several other type I membrane protein substrates including Notch1 and N-cadherin. γ-Secretase is made of four integral transmembrane protein subunits: presenilin (PS), nicastrin, APH1, and PEN2. Multiple lines of evidence indicate that a heteromer of PS-derived N- and C-terminal fragments functions as the catalytic subunit of γ-secretase. Only limited information is available on the domains within each subunit involved in the recognition and recruitment of diverse substrates and the transfer of substrates to the catalytic site. Here, we performed mutagenesis of two domains of PS1, namely the first luminal loop domain (LL1) and the second transmembrane domain (TM2), and analyzed PS1 endoproteolysis as well as the catalytic activities of PS1 toward APP, Notch, and N-cadherin. Our results show that distinct residues within LL1 and TM2 domains as well as the length of the LL1 domain are critical for PS1 endoproteolysis, but not for PS1 complex formation with nicastrin, APH1, and PEN2. Furthermore, our experimental PS1 mutants formed γ-secretase complexes with distinct catalytic properties toward the three substrates examined in this study; however, the mutations did not affect PS1 interaction with the substrates. We conclude that the N-terminal LL1 and TM2 domains are critical for PS1 endoproteolysis and the coordination between the putative substrate-docking site and the catalytic core of the γ-secretase.

FIGURE 1.

The amino acid sequences of PS1 LL1 variants. The sequence of the luminal loop connecting TM domains 1 and 2 is depicted along with the sequences of the LL1 mutants. The aa substitutions are underlined. The shaded letters represent a 12-aa insert. The length of the luminal loop domain (number of aa) is indicated for each construct.

FIGURE 2.

Characterization of γ-secretase complex formation by PS1 LL1 variants. A and B, PS1^−/−/PS2^−/− MEF were stably transduced with recombinant retroviruses expressing the indicated mutants, and the cell lysates were subject to immunoblotting using antibodies against PS1 NTF, PS1 CTF, nicastrin, APH1aL, and PEN2. The levels of GRP78 were analyzed as a loading control. Ins, LL1Ins mutant; imm, immature. C, co-immunoprecipitation (co-IP) analysis of γ-secretase subunits. Stable PS1^−/−/PS2^−/− MEF pools expressing PS1 LL1 variants were lysed in a buffer containing 1% CHAPSO and immunoprecipitated with αPS1Loop or PS1_NT antibody. An aliquot of the input lysate was also analyzed in parallel. The blots were sequentially probed with antibodies against each γ-secretase subunit (PS1_NT, αPS1Loop, nicastrin, APH1aL, and PEN2). The asterisks indicate nonspecific reactivity.

FIGURE 3.

γ-Secretase processing of different substrates by PS1 LL1 mutants. A, stable PS1^−/−/PS2^−/− MEF pools expressing PS1 LL1 variants were transiently infected with APP_Swe retrovirus. The cell lysates were analyzed by immunoblotting with antibody CTM1 (raised against the APP C terminus). The levels of secreted Aβ₄₀ peptides in the conditioned media were quantified by ELISA. Ins, LL1Ins mutant; imm, immature. B, stable PS1^−/−/PS2^−/− MEF pools expressing PS1 LL1 variants were transiently infected with a retrovirus expressing APP C99–6Myc (top panel) or NotchΔEMV-6myc (middle panel). The lysates were analyzed by immunoblotting with mAb 9E10 or N-cadherin antibody. C, co-immunoprecipitation (co-IP) of γ-secretase and APP CTF. Stable PS1^−/−/PS2^−/− MEF pools expressing PS1 LL1 variants were transiently infected with APP_Swe, and the lysates were analyzed by co-immunoprecipitation with PS1_NT antibody and blotted APP antibody. D, co-immunoprecipitation of γ-secretase with NotchΔEMV-6myc and N-cadherin CTF. Lysates of NotchΔEMV-6myc infected or uninfected stable PS1^−/−/PS2^−/− MEF pools were analyzed by co-immunoprecipitation with PS1_NT antibody and blotted with mAb 9E10 and N-cadherin antibody.

FIGURE 4.

PS1 endoproteolysis and the formation of γ-secretase complex in PS1 TM2 variants. A, the aa sequence of the PS1 TM2 domain is depicted along with the corresponding sequence of each PS1 TM2 mutant. The aa substitutions are underlined. B, lysates of PS1^−/−/PS2^−/− MEF stably transduced with retroviruses expressing WT PS1 or the indicated TM2 mutants were analyzed by immunoblotting with antibodies against PS1, nicastrin, APH1aL, and PEN2. The levels of GRP78 were analyzed as a loading control. C, co-immunoprecipitation (co-IP) analysis of γ-secretase subunits was performed essentially as described under the legends to Fig. 2. The asterisks indicate nonspecific reactivity.

FIGURE 5.

γ-Secretase processing of different substrates by PS1 TM2 variants. A, stable PS1^−/−/PS2^−/− MEF pools expressing PS1 TM2 variants were transiently infected with APP_Swe retrovirus. The cell lysates were analyzed by immunoblotting with antibody CTM1. The levels of secreted Aβ₄₀ peptides in the conditioned media were quantified by ELISA. B, stable PS1^−/−/PS2^−/− MEF pools expressing PS1 TM2 variants were transiently infected with a retrovirus expressing APP C99–6Myc (top panel) or NotchΔEMV-6myc (middle panel). The lysates were analyzed by immunoblotting with mAb 9E10. The blots were reprobed with N-cadherin antibody (bottom panel). C, co-immunoprecipitation (co-IP) of γ-secretase and substrates. Stable PS1^−/−/PS2^−/− MEF pools expressing PS1 TM2 variants were transiently infected with retrovirus expressing APP_Swe or NotchΔEMV-6myc. The lysates were analyzed by co-immunoprecipitation with PS1_NT antibody and immunoblotted with APP, 9E10, or N-cadherin antibodies.

FIGURE 6.

Increase of Aβ₄₂ generation by substitutions within TM2. A, the sequence of TM2 domain of WT PS1, WT PS2, and TM2 aa6/15 variant are shown. FAD-linked mutations found within TM2 domain are indicated. Substitutions in aa6/15 variant are underlined. B, stable PS1^−/−/PS2^−/− MEF pools expressing PS1 TM2 variants were transiently infected with a retrovirus expressing APP_Swe. Conditioned medium was collected 48 h after infection, and the levels of secreted Aβ₄₀ and Aβ₄₂ peptides were measured by ELISA.