The Mechanism of γ-Secretase Activities through High Molecular Weight Complex Formation of Presenilins Is Conserved inDrosophila melanogaster and Mammals

Abstract

Mutations in presenilin 1 (PS1) and PS2 genes contribute to the pathogenesis of early onset familial Alzheimer's disease by increasing secretion of the pathologically relevant A␤42 polypeptides. PS genes are also implicated in Notch signaling through proteolytic processing of the Notch receptor in Caenorhabditis elegans, Drosophila melanogaster, and mammals. Here we show that Drosophila PS (Psn) protein undergoes endoproteolytic cleavage and forms a stable high molecular weight (HMW) complex in Drosophila S2 or mouse neuro2a (N2a) cells in a similar manner to mammalian PS. The loss-of-function recessive point mutations located in the C-terminal region of Psn, that cause an early pupal-lethal phenotype resembling Notch mutant in vivo, disrupted the HMW complex formation, and abolished ␥-secretase activities in cultured cells. The overexpression of Psn in mouse embryonic fibroblasts lacking PS1 and PS2 genes rescued the Notch processing. Moreover, disruption of the expression of Psn by double-stranded RNA-mediated interference completely abolished the ␥-secretase activity in S2 cells. Surprisingly, ␥-secretase activity dependent on wild-type Psn was associated with a drastic overproduction of A␤1-42 from human ␤APP in N2a cells, but not in S2 cells. Our data suggest that the mechanism of ␥-secretase activities through formation of HMW PS complex, as well as its abolition by loss-of-function mutations located in the C terminus, are highly conserved features in Drosophila and mammals. Mutations in presenilin (PS1)1 or PS2 genes account for the majority of early-onset familial Alzheimer's disease (FAD), and these mutations cause an increase in the ratio or levels of production of amyloid ␤ peptides ending at position 42 (A␤42), that most readily form amyloid deposits (1). Presenilins are polytopic integral membrane proteins that span the membrane eight times and undergo endoproteolysis (2). The endoproteolytic fragments of PS are incorporated into a high molecular weight (HMW) complex (3, 4) and are highly stabilized (t1 ⁄2 ϭ ϳ20 h), whereas holoprotein is rapidly degraded (t1 ⁄2 ϭ ϳ2 h) (5).PS is implicated in ␥-cleavage of ␤APP, the final step in the generation of A␤ peptides, as well as in the ␥-cleavage-like intramembranous proteolysis of various transmembrane proteins (e.g. Notch, ErbB4, E-cadherin, and LRP) (reviewed in Ref. 6). Although the precise role of PS in the intramembranous proteolysis still remains unknown, following lines of evidence suggest that PS is a catalytic component of ␥-secretase. First, the ablation of PS genes in mice inactivated the total ␥-secretase activities (7,8). Second, mutating either of the two conserved aspartate residues within the transmembrane domains (TMD) 6 and 7 of PS inhibited the ␥-secretase activities (9). Third, the ␥-secretase activity solubilized by a mild detergent, CHAPSO, was immunoprecipitated by antibodies against PS1 in HMW fractions (10). Lastly, the transition-state analogue ␥-secretase inhibitors that are conjugated with photoaffinity labeling and/or biotin t...

Three-dimensional Structure of the Signal Peptide Peptidase

“…Rabbit polyclonal antibody anti-GDN1 against N-terminal region of Drosophila presenilin (Psn) was previously described (21). Rabbit polyclonal antibody SPP CT against the C terminus (358 -377) of human SPP was kindly provided by Dr. Todd Golde (University of Florida) (22).…”

“…Recombinant baculovirus was generated according to the manufacturer's instructions. Maintenance and transfection of S2 and Sf9 cells were performed as described previously (11,21,27,28). Sf9 cells (2 ϫ 10 6 cells/ml) were infected with a combination of recombinant baculoviruses at the virus volume of maximum expression level and harvested 72 h after infection.…”

Nicastrin Is Dispensable for γ-Secretase Protease Activity in the Presence of Specific Presenilin Mutations

“…To test whether the PS1 F411Y/ S438P mutant forms a complex with other components, ␥-secretase was immunopurified from yeast microsomes with the PS1 antibody against loop region (G1L3) (42) (Fig. 6A).…”

Sulindac Sulfide Is a Noncompetitive γ-Secretase Inhibitor That Preferentially Reduces Aβ42 Generation

“…A cDNA encoding C100-FmH carrying I716F mutation (11,27) was generated by the long PCR protocol using a cDNA encoding C100-FLAG in pTrcHis2A vector as a template. All constructs were sequenced using Thermo Sequenase TM (Amersham Biosciences) on an automated sequencer (Li-Cor).…”