The first proline of PALP motif at the C terminus of presenilins is obligatory for stabilization, complex formation, and gamma-secretase activities of presenilins.

Taisuke Tomita,Yuichi Morohashi,Tomonari Watabiki,Takeshi Iwatsubo,Bart De Strooper,Nobumasa Takasugi,Rie Takikawa,Raphael Kopan

doi:10.1074/jbc.m011152200

Abstract

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer's disease by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). PS is an evolutionarily conserved multipass transmembrane protein, and all known PS proteins contain a proline-alanine-leucine-proline (PALP) motif starting at proline (P) 414 (amino acid numbering based on human PS2) at the C terminus. Furthermore, missense mutations that replace the first proline of PALP with leucine (P414L) lead to a loss-of-function of PS in Drosophila melanogaster and Caenorhabditis elegans. To elucidate the roles of the PALP motif in PS structure and function, we analyzed neuro2a as well as PS1/2 null fibroblast cell lines transfected with human PS harboring mutations at the PALP motif. P414L mutation in PS2 (and its equivalent in PS1) abrogated stabilization, high molecular weight complex formation, and entry to Golgi/trans-Golgi network of PS proteins, resulting in failure of Abeta42 overproduction on familial Alzheimer's disease mutant basis as well as of site-3 cleavage of Notch. These data suggest that the first proline of the PALP motif plays a crucial role in the stabilization and formation of the high molecular weight complex of PS, the latter being the active form with intramembrane proteolytic activities.

Highlights

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer’s disease by increasing production of the amyloidogenic form of amyloid ␤ peptides ending at residue 42 (A␤42)
Senile plaques are composed of amyloid ␤ peptides (A␤) comprising ϳ40 amino acids that are proteolytically produced from ␤-amyloid precursor protein (␤APP). ␤APP is initially cleaved by ␤-secretase to generate a 99-residue C-terminal fragment (C99) that is cleaved by ␥-secretase to generate A␤
Immunoblot analysis of the cell lysates showed that neither PS2/D366A nor PS2/⌬loopN underwent endoproteolysis to give rise to a 35-kDa NTF and a 23-kDa CTF that are normally produced from full-length (FL) PS2 as reported previously (Fig. 2A, upper panel, arrow) [13, 14, 25,26,27]. wt and mt PS2/P414L were expressed as holoproteins and did not yield endoproteolytic fragments, whereas PS2/P417S were cleaved to form fragments (Fig. 2A, upper panel, arrowheads)

Summary

Introduction

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer’s disease by increasing production of the amyloidogenic form of amyloid ␤ peptides ending at residue 42 (A␤42). Studies in Caenorhabditis elegans and Drosophila melanogaster, as well as in knockout mice, suggested that PS facilitates Notch signaling by activating the ligand-induced intramembranous proteolysis of Notch receptors at site-3 to release their cytoplasmic domains (NICD) [7,8,9,10,11] This cleavage appears to be very similar to ␥-cleavage of ␤APP because it occurs close to or within the membrane, inhibited by inactivation of PS genes, and can be blocked by peptidomimetic ␥-secretase inhibitors (reviewed in Ref. 12). The facts that the amount of stabilized NTF and CTF of PS are tightly regulated and that overexpression of exogenous PS replaces endogenous PS suggest that some cellular factor(s) of limited amount are required for the stabilization and complex formation of PS [19, 30]

Methods

Results

Conclusion