Three-Amino Acid Spacing of Presenilin Endoproteolysis Suggests a General Stepwise Cleavage of  -Secretase-Mediated Intramembrane Proteolysis

Abstract

Presenilin (PS1 or PS2) is the catalytic component of the ␥-secretase complex, which mediates the final proteolytic processing step leading to the Alzheimer's disease (AD)-characterizing amyloid ␤-peptide. PS is cleaved during complex assembly into its characteristic N-and C-terminal fragments. Both fragments are integral components of physiologically active ␥-secretase and harbor the two critical aspartyl residues of the active site domain. While the minimal subunit composition of ␥-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 ␥-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 -, -, and ␥-cleavages described for typical ␥-secretase substrates, such as the ␤-amyloid precursor protein. Our findings therefore suggest that intramembranous cleavage by ␥-secretase and related intramembrane-cleaving proteases may generally occur via stepwise endoproteolysis.

Lysine 624 of the Amyloid Precursor Protein (APP) Is a Critical Determinant of Amyloid β Peptide Length

“…1A) (34). Our finding that N-terminal mutations outside the transmembrane domain of APP can cause dramatic shifts (up to seven residues) in the final site of the ␥ cut, without any significant change at the ⑀ site, support the idea that ⑀ cleavage occurs first, followed by sequential proteolysis, as has been proposed by a number of groups (8,35,36). Although we favor this sequential cleavage model of ␥-secretase activity, one unexplained phenomenon is the C-terminal heterogeneity in the final site of ␥ cleavage to produce A␤ and analogous peptides from other substrates (5).…”

Presenilin Transmembrane Domain 8 Conserved AXXXAXXXG Motifs Are Required for the Activity of the γ-Secretase Complex

“…In other terms, and although the stepwise process of ␥-secretase assembly is not fully understood, NCT is extensively glycosylated once the four core subunits (PS, APH-1, PEN-2, and NCT) are assembled (14). ␥-Secretase activation occurs when PS undergoes endoproteolysis, leading to the release from the active site of the inhibitory cytosolic loop, thereby activating the enzyme (66,77). When expressed with the other ␥-secretase components, all PS forms, including the PS-AA and PS-AG mutants, were able to generate a fully assembled ␥-secretase.…”

Nicastrin functions to sterically hinder γ-secretase–substrate interactions driven by substrate transmembrane domain

“…and 7 of its nine TMDs (17,25,26). After initial complex formation, the mature proteolytically active complex is formed when presenilin undergoes auto-proteolysis, resulting in N-and C-terminal fragments (NTF and CTF, respectively) (17,27,28), a process thought to be stimulated by the three-TMD component Pen-2 (29). The seven-TMD protein Aph-1 is believed to play a scaffolding role in complex formation (30,31).…”