Mutations in human presenilin (PS) genes cause aggressive forms of familial Alzheimer's disease. Presenilins are polytopic proteins that harbour the catalytic site of the c-secretase complex and cleave many type I transmembrane proteins including b-amyloid precursor protein (APP), Notch and syndecan 3. Contradictory results have been published concerning whether PS mutations cause 'abnormal' gain or (partial) loss of function of c-secretase. To avoid the possibility that wild-type PS confounds the interpretation of the results, we used presenilin-deficient cells to analyse the effects of different clinical mutations on APP, Notch, syndecan 3 and N-cadherin substrate processing, and on c-secretase complex formation. A loss in APP and Notch substrate processing at e and S3 cleavage sites was observed with all presenilin mutants, whereas APP processing at the c site was affected in variable ways. PS1-D9 and PS1-L166P mutations caused a reduction in b-amyloid peptide (Ab) 40 production whereas PS1-G384A mutant significantly increased Ab 42 . Interestingly PS2, a close homologue of PS1, appeared to be a less efficient producer of Ab than PS1. Finally, subtle differences in c-secretase complex assembly were observed. Overall, our results indicate that the different mutations in PS affect c-secretase structure or function in multiple ways.

γ‐Secretase‐dependent regulated intramembrane proteolysis of amyloid precursor protein (APP) releases the APP intracellular domain (AICD). The question of whether this domain, like the Notch intracellular domain, is involved in nuclear signalling is highly controversial. Although some reports suggest that AICD regulates the expression of KAI1, glycogen synthase kinase‐3β, Neprilysin and APP, we found no consistent effects of γ‐secretase inhibitors or of genetic deficiencies in the γ‐secretase complex or the APP family on the expression levels of these genes in cells and tissues. Finally, we demonstrate that Fe65, an important AICD‐binding protein, transactivates a wide variety of different promoters, including the viral simian virus 40 promoter, independent of AICD coexpression. Overall, the four currently proposed target genes are at best indirectly and weakly influenced by APP processing. Therefore, inhibition of APP processing to decrease Aβ generation in Alzheimer's disease will not interfere significantly with the function of these genes.

␥-Secretase is a multiprotein complex responsible for the intramembranous cleavage of the amyloid precursor protein and other type I transmembrane proteins. Mutations in Presenilin, the catalytic core of this complex, cause Alzheimer disease. Little is known about the structure of the protein and even less about the catalytic mechanism, which involves proteolytic cleavage in the hydrophobic environment of the cell membrane. It is basically unclear how water, needed to perform hydrolysis, is provided to this reaction. Presenilin transmembrane domains 6 and 7 seem critical in this regard, as each bears a critical aspartate contributing to catalytic activity. Current models imply that both aspartyl groups should closely oppose each other and have access to water. This is, however, still to be experimentally verified. Here, we have performed cysteine-scanning mutagenesis of both domains and have demonstrated that several of the introduced residues are exposed to water, providing experimental evidence for the existence of a water-filled cavity in the catalytic core of Presenilin. In addition, we have demonstrated that the two aspartates reside within this cavity and are opposed to each other in the native complex. We have also identified the conserved tyrosine 389 as a critical partner in the catalytic mechanism. Several additional amino acid substitutions affect differentially the processing of ␥-secretase substrates, implying that they contribute to enzyme specificity. Our data suggest the possibility that more selective ␥-secretase inhibitors could be designed. The Presenilin (PS)2 proteins are the prototypic members of a group of aspartic proteases involved in regulated intramembrane proteolysis, a mechanism responsible for cleavage of peptide bonds within the lipid bilayer (1, 2). More than 150 mutations in Presenilin 1 and 10 mutations in Presenilin 2 have been associated with Alzheimer disease (for a list of the mutations, see molgen.ua.ac.be/ADMutations), demonstrating their pivotal role in the pathogenesis of the disease. Presenilins are critical for the ␥-secretase cleavage of the amyloid precursor protein (APP) that generates the amyloid ␤ peptide (A␤) (3). They are also responsible for the intramembrane proteolysis of several other type I transmembrane proteins (reviewed in Ref. 4), including the S3 cleavage of Notch that releases the Notch intracellular domain (NICD), a major regulator of gene transcription (5). Together with Presenilin, three other membrane proteins are necessary and sufficient for processing by ␥-secretase (6 -8), i.e. Nicastrin, APH-1, and PEN-2. Nicastrin appears to recognize the free N terminus of potential substrates (9), whereas the catalytic core resides in Presenilin (reviewed in Ref. 10).Most recent topological studies propose a nine-transmembrane domain model for Presenilins, with the N terminus oriented toward the cytosol and the C terminus toward the extracellular space (11-13). Mutation of two conserved aspartates, Asp-257 and Asp-385, located in transmembrane domains (TMs)...

One of the most prominent drug targets for the treatment of Alzheimer disease is ␥-secretase, a multi-protein complex responsible for the generation of the amyloid-␤ peptide. The catalytic core of the complex lies on presenilin, a multi-spanning membrane protease, the activity of which depends on two aspartate residues located in transmembrane domains 6 and 7. We have recently shown by cysteine-scanning mutagenesis that these aspartates are facing a water-filled cavity in the lipid bilayer, demonstrating how proteolytic cleavage of the substrates can be taking place within the membrane. Here, we demonstrate that transmembrane domain 9 and hydrophobic domain VII in the large cytoplasmic loop of presenilin are dynamic structural parts of this cavity. Hydrophobic domain VII is associated with transmembrane domain 7 in the membrane, probably facilitating the entrance of water molecules in the catalytic site. Transmembrane domain 9, on the other hand, exhibits a highly flexible structure, potentially involved in the transport of substrates to the catalytic site, as well as in the binding of ␥-secretase inhibitors. The conserved proline-alanineleucine motif at the cytoplasmic part of this domain is extremely close to the catalytic Asp 257 and is crucial for conformational changes leading to the activation of the catalytic site. We, also, identify a unique mutant in this domain (I437C) that specifically blocks amyloid-␤ peptide production without affecting the processing of the physiologically indispensable Notch substrate. Our data are finally combined to propose a model for the architectural organization and activation of the catalytic site of presenilin.