Presenilin-1 (PS1) facilitates ␥-secretase cleavage of the ␤-amyloid precursor protein and the intramembraneous cleavage of Notch1. Although Alzheimer's diseaseassociated mutations in the homologous presenilin (PS2) gene elevate amyloid ␤-peptide (A␤42) production like PS1 mutations, here we demonstrate that a gene ablation of PS2 (unlike that of PS1) in mice does not result in a severe phenotype resembling that of Notchablated animals. To investigate the amyloidogenic function of PS2 more directly, we mutagenized a conserved aspartate at position 366 to alanine, because the corresponding residue of PS1 is known to be required for its amyloidogenic function. Cells expressing the PS2 D366A mutation exhibit significant deficits in proteolytic processing of ␤-amyloid precursor protein indicating a defect in ␥-secretase activity. The reduced ␥-secretase activity results in the almost complete inhibition of A␤ and p3 production in cells stably expressing PS2 D366A, whereas cells overexpressing the wild-type PS2 cDNA produce robust levels of A␤ and p3. Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation not only blocks ␥-secretase activity but also inactivates PS2 activity in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain. These data suggest that PS2 is functionally involved in A␤ production and Notch signaling by facilitating similar proteolytic cleavages.

Endoproteolysis of beta-amyloid precursor protein (betaAPP) and Notch requires conserved aspartate residues in presenilins 1 and 2 (PS1 and PS2). Although PS1 and PS2 have therefore been proposed to be aspartyl proteases, no homology to other aspartyl proteases has been found. Here we identify homology between the presenilin active site and polytopic aspartyl proteases of bacterial origin, thus supporting the hypothesis that presenilins are novel aspartyl proteases.

We identified four proteins in nuclear extracts from HeLa cells which specifically bind to a scaffold attachment region (SAR) element from the human genome. Of these four proteins, SAF‐A (scaffold attachment factor A), shows the highest affinity for several homologous and heterologous SAR elements from vertebrate cells. SAF‐A is an abundant nuclear protein and a constituent of the nuclear matrix and scaffold. The homogeneously purified protein is a novel double stranded DNA binding protein with an apparent molecular weight of 120 kDa. SAF‐A binds at multiple sites to the human SAR element; competition studies with synthetic polynucleotides indicate that these sites most probably reside in the multitude of A/T‐stretches which are distributed throughout this element. In addition we show by electron microscopy that the protein forms large aggregates and mediates the formation of looped DNA structures.

Numerous mutations causing early onset Alzheimer's disease have been identified in the presenilin (PS) genes, particularly the PS1 gene. Like the mutations identified within the ␤-amyloid precursor protein gene, PS mutations cause the increased generation of a highly neurotoxic variant of amyloid ␤-peptide. PS proteins are proteolytically processed to an N-terminal ϳ30-kDa (NTF) and a C-terminal ϳ20-kDa fragment (CTF 20 ) that form a heterodimeric complex. We demonstrate that this complex is resistant to proteolytic degradation, whereas the full-length precursor is rapidly degraded. Degradation of the PS1 holoprotein is sensitive to inhibitors of the proteasome. Formation of a heterodimeric complex is required for the stability of both PS1 fragments, since fragments that do not co-immunoprecipitate with the PS complex are rapidly degraded by the proteasome. Mutant PS fragments not incorporated into the heterodimeric complex lose their pathological activity in abnormal amyloid ␤-peptide generation even after inhibition of their proteolytic degradation. The PS1 heterodimeric complex can be attacked by proteinases of the caspase superfamily that generate an ϳ10-kDa proteolytic fragment (CTF 10 ) from CTF 20 . CTF 10 is rapidly degraded most likely by a calpain-like cysteine proteinase. From these data we conclude that PS1 metabolism is highly controlled by multiple proteolytic activities indicating that subtle changes in fragment generation/ degradation might be important for Alzheimer's diseaseassociated pathology.Alzheimer's disease (AD) 1 is the most common dementia worldwide. A pathological hallmark of AD is the invariant accumulation of numerous senile plaques in certain areas of the brain. Senile plaques are composed of the amyloid ␤-peptide (A␤), a proteolytic derivative of the ␤-amyloid precursor protein (␤APP; see Ref. 1). In the majority of cases, AD occurs as a sporadic disease with an increasing risk during aging (2). However, in about 10 -15% of the cases AD is caused by autosomal dominant mutations within three genes (2). A very limited set of families was identified carrying mutations in the ␤APP gene (2). These mutations occur at, or close to, the cleavage sites of the ␤APP-processing enzymes, the secretases (1). All ␤APP mutations analyzed so far cause an enhanced production of A␤, specifically the highly pathogenic 42-amino acid variant, A␤42 (2).By far the highest number of FAD-associated mutations were identified within the PS gene (see Ref. 3 and for review see Ref. 4). Two mutations were also observed in the highly homologous PS2 gene (5, 6). PS proteins are integral membrane proteins, which form 6 to 8 trans-membrane domains with the N-terminal domain, the large loop, and the C-terminal domain located within the cytoplasm (7,8). Both PS proteins are predominantly expressed within the endoplasmic reticulum, the nuclear envelope, and the early Golgi (7, 9 -12).Like ␤APP mutations, mutant PS proteins are involved in aberrant A␤ generation. All PS1 and PS2 mutations analyzed so far affect the ...

The two homologous presenilins are key factors for the generation of amyloid ␤-peptide (A␤), since Alzheimer's disease (AD)-associated mutations enhance the production of the pathologically relevant 42-amino acid A␤ (A␤42), and a gene knockout of presenilin-1 (PS1) significantly inhibits total A␤ production. Presenilins undergo proteolytic processing within the domain encoded by exon 9, a process that may be closely related to their biological and pathological activity. An AD-associated mutation within the PS1 gene deletes exon 9 (PS1⌬exon9) due to a splicing error and results in the accumulation of the uncleaved full-length protein. We now demonstrate the unexpected finding that the pathological activity of PS1⌬exon9 is independent of its lack to undergo proteolytic processing, but is rather due to a point mutation (S290C) occurring at the aberrant exon 8/10 splice junction. Mutagenizing the cysteine residue at position 290 to the original serine residue completely inhibits the pathological activity in regard to the elevated production of A␤42. Like PS1⌬exon9, the resulting presenilin variant (PS1⌬exon9 C290S) accumulates as an uncleaved protein and fully replaces endogenous presenilin fragments. Moreover, PS1⌬exon9 C290S exhibits a significantly increased biological activity in a highly sensitive in vivo assay as compared with the ADassociated mutation. Therefore not only the increased A␤42 production but also the decreased biological function of PS1⌬exon9 is due to a point mutation and independent of the lack of proteolytic processing.