Alexandra Tolia

Presenilin clinical mutations can affect gamma-secretase activity by different mechanisms.

Mutations in human presenilin (PS) genes cause aggressive forms of familial Alzheimers disease. Presenilins are polytopic proteins that harbour the catalytic site of the γ‐secretase complex and cleave many type I transmembrane proteins including β‐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 γ‐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 γ‐secretase complex formation. A loss in APP and Notch substrate processing at ɛ and S3 cleavage sites was observed with all presenilin mutants, whereas APP processing at the γ site was affected in variable ways. PS1‐Δ9 and PS1‐L166P mutations caused a reduction in β‐amyloid peptide (Aβ)40 production whereas PS1‐G384A mutant significantly increased Aβ42. Interestingly PS2, a close homologue of PS1, appeared to be a less efficient producer of Aβ than PS1. Finally, subtle differences in γ‐secretase complex assembly were observed. Overall, our results indicate that the different mutations in PS affect γ‐secretase structure or function in multiple ways.

Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes

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 Alzheimers disease will not interfere significantly with the function of these genes.

Contribution of presenilin transmembrane domains 6 and 7 to a water-containing cavity in the gamma-secretase complex

γ-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 γ-secretase substrates, implying that they contribute to enzyme specificity. Our data suggest the possibility that more selective γ-secretase inhibitors could be designed.

Presenilin-1 maintains a nine transmembrane topology throughout the secretory pathway

Presenilin-1 is a polytopic membrane protein that assembles with nicastrin, PEN-2, and APH-1 into an active γ-secretase complex required for intramembrane proteolysis of type I transmembrane proteins. Although essential for a correct understanding of structure-function relationships, its exact topology remains an issue of strong controversy. We revisited presenilin-1 topology by inserting glycosylation consensus sequences in human PS1 and expressing the obtained mutants in a presenilin-1 and 2 knock-out background. Based on the glycosylation status of these variants we provide evidence that presenilin-1 traffics through the Golgi after a conformational change induced by complex assembly. Based on our glycosylation variants of presenilin-1 we hypothesize that complex assembly occurs during transport between the endoplasmic reticulum and the Golgi apparatus. Furthermore, our data indicate that presenilin-1 has a nine-transmembrane domain topology with the COOH terminus exposed to the lumen/extracellular surface. This topology is independently underscored by lysine mutagenesis, cell surface biotinylation, and cysteine derivation strategies and is compatible with the different physiological functions assigned to presenilin-1.

Glu332 in the Nicastrin Ectodomain Is Essential for γ-Secretase Complex Maturation but Not for Its Activity

The γ-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin has been proposed to operate as the substrate receptor of the complex with the glutamate 332 (Glu333 in human) serving as the anionic binding site for the α-amino-terminal group of substrates. The putative binding site is located within the aminopeptidase-like domain of Nicastrin. The Glu332 is proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu332 could bind the α-amino-terminal group of substrates, we hypothesized, in analogy with M28-aminopeptidases, that other residues in the putative binding site of Nicastrin should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of APP and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolishes the anionic α-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased γ-secretase complex levels, but the remaining complex appeared as active as the wild-type complex. We confirmed in two independent in vitro assays that the specific enzymatic activity of the E332A mutant was comparable with that of the wild-type complex. Thus, Glu332 crucially affects complex maturation rather than substrate recognition. Moreover other Nicastrin mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally γ-secretase activity. Consequently, these studies indicate that the main role of the Glu332 is in the maturation and assembly of γ-secretase rather than in the recognition of the substrates.

Structure and function of gamma-secretase.

The gamma-secretase complex is a prime target for pharmacological intervention in Alzheimers disease and so far drug discovery efforts have yielded a large variety of potent and rather specific inhibitors of this enzymatic activity. However, as gamma-secretase is able to cleave a wide variety of physiological important substrates, the real challenge is to develop substrate-specific compounds. Therefore, obtaining structural information about gamma-secretase is indispensable. As crystal structures of the complex will be difficult to achieve, applied biochemical approaches need to be integrated with structural information obtained from other intramembrane-cleaving proteases. Here we review current knowledge about the structure and function of gamma-secretase and discuss the value of these findings for the mechanistic understanding of this unusual protease.

Transmembrane domain 9 of presenilin determines the dynamic conformation of the catalytic site of gamma-secretase.

One of the most prominent drug targets for the treatment of Alzheimer disease is γ-secretase, a multi-protein complex responsible for the generation of the amyloid-β 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 γ-secretase inhibitors. The conserved proline-alanine-leucine motif at the cytoplasmic part of this domain is extremely close to the catalytic Asp257 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-β 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.

Mutagenesis Mapping of the Presenilin 1 Calcium Leak Conductance Pore

Missense mutations in presenilin 1 (PS1) and presenilin 2 (PS2) proteins are a major cause of familial Alzheimer disease. Presenilins are proteins with nine transmembrane (TM) domains that function as catalytic subunits of the γ-secretase complex responsible for the cleavage of the amyloid precursor protein and other type I transmembrane proteins. The water-filled cavity within presenilin is necessary to mediate the intramembrane proteolysis reaction. Consistent with this idea, cysteine-scanning mutagenesis and NMR studies revealed a number of water-accessible residues within TM7 and TM9 of mouse PS1. In addition to γ-secretase function, presenilins also demonstrate a low conductance endoplasmic reticulum Ca2+ leak function, and many familial Alzheimer disease presenilin mutations impair this function. To map the potential Ca2+ conductance pore in PS1, we systematically evaluated endoplasmic reticulum Ca2+ leak activity supported by a series of cysteine point mutants in TM6, TM7, and TM9 of mouse PS1. The results indicate that TM7 and TM9, but not TM6, could play an important role in forming the conductance pore of PS1. These results are consistent with previous cysteine-scanning mutagenesis and NMR analyses of PS1 and provide further support for our hypothesis that the hydrophilic catalytic cavity of presenilins may also constitute a Ca2+ conductance pore.

Functional and topological analysis of Pen-2, the fourth subunit of the gamma-secretase complex

The γ-secretase complex is a member of the family of intramembrane cleaving proteases, involved in the generation of the Aβ peptides in Alzheimer disease. One of the four subunits of the complex, presenilin, harbors the catalytic site, although the role of the other three subunits is less well understood. Here, we studied the role of the smallest subunit, Pen-2, in vivo and in vitro. We found a profound Notch-deficiency phenotype in Pen-2−/− embryos confirming the essential role of Pen-2 in the γ-secretase complex. We used Pen-2−/− fibroblasts to investigate the structure-function relation of Pen-2 by the scanning cysteine accessibility method. We showed that glycine 22 and proline 27 in hydrophobic domain 1 of Pen-2 are essential for complex formation and stability of γ-secretase. We also demonstrated that hydrophobic domain 1 and the loop domain of Pen-2 are located in a water-containing cavity and are in short proximity to the presenilin C-terminal fragment. We finally demonstrated the essential role of Pen-2 for the proteolytic activity of the complex. Our study supports the hypothesis that Pen-2 is more than a structural component of the γ-secretase complex and may contribute to the catalytic mechanism of the enzyme.

P3-398: Cysteine scanning mutagenesis as a tool for structure-function analysis of presenilin 1

the active site domain located in its catalytic subunit presenilin (PS), where an additional substrate binding site has been proposed. Objective: To identify sequence determinants in the PS active site domain possibly involved in -secretase substrate identification. To further characterize the PS active site domain, in particular the role of the conserved GxGD protease active site motif. Methods: Mutants of the PS active site domain were generated. The constructs were assessed for -secretase activity towards APP and Notch substrates in embryonic fibroblast cells derived from PS1/2-/knockout mice cells or in HEK293 cells. In addition, the mutants were tested for their rescuing activity of a Notch-signaling deficient C. elegans sel-12 mutant. Results and Conclusions: When the active site domain of PS1 located in transmembrane domains 6 and 7 was exchanged with that of the C. elegans sperm protein SPE-4, the most distant PS homologue, the chimeric protein, PS1/SPE-46/7, supported APP but not Notch processing. In addition, PS1/SPE-46/7 was strongly impaired in C. elegans Notch signaling in vivo. Mapping experiments identified a single amino acid at position (amino acid 383 in PS1) of the GxGD active site motif in transmembrane domain 7 of PS to be responsible for the observed defect in Notch processing and signaling. Our data thus implicate a role of the PS active site domain in APP/Notch substrate selectivity of -secretase. We have also generated mutants of G382 of PS1 to further assess the functional role of the GxGD motif. The progress on these studies will be presented.