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

Abstract

␥-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial ␥-secretase substrates containing amyloid precursor protein or Notch fragments. The ␥-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4p release from the membrane-bound substrates, i.e. growth of yeast on histidine and adenine, or ␤-galactosidase assay. We screened and evaluated ␥-secretase mutants using this reconstitution system in yeast, which does not possess endogenous ␥-secretase activity. When we introduced familial Alzheimer mutants of PS1 in this system, their activities were shown to be loss of function. Although the protease activity of wild type PS1 depends on the other three subunits introduced, we obtained 15 new PS1 mutants, which are active in the absence of NCT. They possessed a S438P mutation at the ninth transmembrane domain (TM9) together with one missense mutation distributed through transmembrane and loop regions. These mutations were not related to familial Alzheimer mutations of PS1 as identified so far. The S438P mutant was partially active but required other mutations for full activation. Results of the ␤-galactosidase assay suggested that they have wild type protease activities, which were further confirmed by the endoproteolysis of PS1, amyloid ␤ peptides, and Notch intracellular domain production in mammalian cells. These results suggest that NCT is dispensable for the protease activity of ␥-secretase.

Contribution of the γ-Secretase Subunits to the Formation of Catalytic Pore of Presenilin 1 Protein

“…This result is consistent with the finding that Nct-Aph-1 interacts with PS1 CTF (25,26,40). Intriguingly, it has been shown that a point mutation at TMD9 rendered PS1 enzymatically active without binding to Nct (41). These results support our notion that the structure of TMD9 was affected by binding of the NctAph-1 subcomplex.…”

Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1

“…40 The activity and ␥-secretase dependence of the Notch1 polypeptide created by translational initiation at M1727 argues against this model and is consistent with other recent genetic studies indicating that nicastrin stabilizes ␥-secretase, but is nonessential for ␥-secretase recognition and cleavage of Notch. 41,42 Although murine T-ALL is commonly associated with 5Ј deletions in Notch1, analysis of close to 200 human T-ALLs (on Affymetrix arrays, with either 615 000 features (250K ϩ 50K arrays) or SNP 6 arrays (1.8 million features) has not revealed focal copy number variants in NOTCH1 (Dr Charles Mullighan, St Jude Children's Research Hospital, written communication, August 4, 2010). It thus appears that the relatively large deletions that are observed frequently in murine Notch1 are rare, if they occur at all, in NOTCH1 in human T-ALL, with the important caveat that additional studies are needed to exclude the presence of smaller deletions or point mutations involving the 5Ј end of the gene.…”

Comparison of Presenilin 1 and Presenilin 2 γ-Secretase Activities Using a Yeast Reconstitution System

“…Construction of ␥-Secretase and Substrates-To reconstitute ␥-secretase in yeast, human PS1 or PS2, NCT, Aph1a-L-HA, FLAG-Pen2, and substrates were cloned into the following vectors, as described previously (15). Briefly, PS1 or PS2 and NCT were ligated into KpnI and XbaI sites of the pBEVY-T vector (16).…”