Activation and intrinsic gamma-secretase activity of presenilin 1

Abstract

A complex composed of presenilin (PS), nicastrin, PEN-2, and APH-1 is absolutely required for γ-secretase activity in vivo. Evidence has emerged to suggest a role for PS as the catalytic subunit of γ-secretase, but it has not been established that PS is catalytically active in the absence of associated subunits. We now report that bacterially synthesized, recombinant PS (rPS) reconstituted into liposomes exhibits γ-secretase activity. Moreover, an rPS mutant that lacks a catalytic aspartate residue neither exhibits reconstituted γ-secretase activity nor interacts with a transition-state γ-secretase inhibitor. Importantly, we demonstrate that rPS harboring mutations that cause early onset familial Alzheimer's disease (FAD) lead to elevations in the ratio of Aβ42 to Aβ40 peptides produced from a wild-type APP substrate and that rPS enhances the Aβ42/Aβ40 peptide ratio from FAD-linked mutant APP substrates, findings that are entirely consistent with the results obtained in in vivo settings. Thus, γ-secretase cleavage specificity is an inherent property of the polypeptide. Finally, we demonstrate that PEN2 is sufficient to promote the endoproteolysis of PS1 to generate the active form of γ-secretase. Thus, we conclusively establish that activated PS is catalytically competent and the bimolecular interaction of PS1 and PEN2 can convert the PS1 zymogen to an active protease.

Fig. 1.

Preparation of proteoliposome containing presenilin exon 9 deletion mutant (PS1ΔE9). (A) Diagram of MBP-fused PS1ΔE9 protein. The PS1 exon 9 (E9) domain contains the endoproteolytic cleavage site and has been deleted to create PS1ΔE9. MBP (maltose-binding protein) has been fused at the N terminus of PS1 for purification. A thrombin cleavage site has been incorporated between MBP and PS1ΔE9 for subsequent MBP removal. (B) Purification and reconstitution procedure for PS1ΔE9 insertion into proteoliposomes. MBP-fused PS1ΔE9 was overexpressed in E. coli and purified using affinity chromatography with an amylose column. The purified MBP-PS1ΔE9 protein was introduced into liposomal membranes with CHAPS detergent and reconstructed as a mixture of unincorporated proteins and proteoliposomes after dialysis. Following thrombin removal of the MBP-domain, the end product PS1ΔE9 proteoliposome was isolated via sucrose density-gradient centrifugation. (C) Coomassie blue staining of purified proteins that were isolated after thrombin cleavage and density-gradient centrifugation.

Fig. 2.

The reconstituted PS1ΔE9-liposomes exhibit γ-secretase activity. (A) The relationship between the amount of PS1ΔE9 protein within proteoliposome and γ-secretase activity (Mean ± SD. n≥3). γ-Secretase activity in proteoliposomes was assayed using biotinylated Sb4 substrate (1 μM) in the presence and absence of L458 inhibitor. The Aβ40 product was detected by electrochemiluminescence (ECL) using G2-10 antibody. (B) JC-8 photolabeling of proteoliposomes containing PS1ΔE9 or PS1ΔE9-D385A. JC-8, a photoactivatable active-site-directed γ-secretase inhibitor (see top panel for its structure) was used to label the reconstituted proteoliposomes in the presence and absence of excess L458 (1 μM). After photolabeling, biotinylated proteins were isolated using streptavidin and then analyzed by Western blotting with anti-PS1-NTF antibody. (C) Schematic representation of microspheres and the PS1-Cpd5- AF633-SA complex and confocal microscopy visualization of the distribution of PS1ΔE9:Cpd 5:AF633-SA complexes on the proteolipobead surface. The 3D-image reconstructions of the distribution of AF633-SA at the surface of proteolipobeads were obtained from the XYZ data subsets. (D) Effect of PS1ΔE9-C290S on rates of γ-secretase for Aβ40 and Aβ42 production and the ratio of Aβ42∶Aβ40. (Mean ± SD. n = 4, differences were evaluated by Student’s t test. p-values: *< 0.05; ** < 0.01).

Fig. 3.

Effect of PS1 and APP FAD mutations on γ-secretase activity and specificity within reconstituted proteoliposomes. (A) Coomassie blue staining of purified PS1ΔE9 mutated proteins (G355A and L166P). All proteoliposomes were isolated by density-gradient centrifugation (see Fig. 1B). (B) Effect of PS1ΔE9 mutations on γ-secretase activity for the Aβ40 and Aβ42 site cleavages. (C) Effect of PS1ΔE9 mutations on the ratio of Aβ42∶Aβ40. (D) Effect of APP mutations on γ-secretase activity. (E) Effect of APP mutations on the ratio of Aβ42∶Aβ40. (Mean ± SD. n = 4, differences were evaluated by Student’s t test p-values: * < 0.05; ** < 0.01; *** < 0.001).

Fig. 4.

Proteoliposome containing both full-length PS1 and PEN2 proteins exhibits γ-secretase activity. (A) Western analysis with PS1 NTF and PEN2 antibodies. Proteoliposome containing both PS1 and PEN2 shows a specific band corresponding to PS1 NTF. * Breakdown product of unknown identity. (B) Proteoliposome containing both PS1 and PEN2 displayed γ-secretase activity. γ-Secretase activity was measured by G2-10 antibody that recognizes with Aβ40 with AlphaLISA technology. (C) The PS1-NTF was specifically labeled by JC-8, but not PS1-FL in the PS1 + PEN2 liposome. Furthermore, this labeling was blocked by L458.

Fig. 5.

The proposed activation mechanism of PS1. PS1-FL is considered as a zymogen for proteolysis and active-site-directed inhibitor binding. Endoproteolysis and deletion at exon 9 convert PS1-FL into the active enzyme. Exon 9 can serve a steric constraint for positioning the two catalytic Asp residues.