Abeta42 overproduction associated with structural changes in the catalytic pore of gamma-secretase: common effects of Pen-2 N-terminal elongation and fenofibrate

gamma-Secretase is an atypical aspartyl protease that cleaves amyloid beta-precursor protein to generate Abeta peptides that are causative for Alzheimer disease. gamma-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1, and Pen-2. Pen-2 directly binds to transmembrane domain 4 of PS and confers proteolytic activity on gamma-secretase, although the mechanism of activation and its role in catalysis remain unknown. Here we show that an addition of amino acid residues to the N terminus of Pen-2 specifically increases the generation of Abeta42, the longer and more aggregable species of Abeta. The effect of the N-terminal elongation of Pen-2 on Abeta42 generation was independent of the amino acid sequences, the expression system and the presenilin species. In vitro gamma-secretase assay revealed that Pen-2 directly affects the Abeta42-generating activity of gamma-secretase. The elongation of Pen-2 N terminus caused a reduction in the water accessibility of the luminal side of the catalytic pore of PS1 in a similar manner to that caused by an Abeta42-raising gamma-secretase modulator, fenofibrate, as determined by substituted cysteine accessibility method. These data suggest a unique mechanism of Abeta42 overproduction associated with structural changes in the catalytic pore of presenilins caused commonly by the N-terminal elongation of Pen-2 and fenofibrate.     

FAD-linked mutations in presenilin 1 alter the length of Abeta peptides derived from betaAPP transmembrane domain mutants

gamma-Secretase is an enzymatic activity responsible for the final cleavage of the amyloid precursor protein leading to the production of the amyloid beta-peptide (Abeta). gamma-Secretase is likely an aspartyl protease, since its activity can be inhibited by both pepstatin and active-site directed aspartyl protease inhibitors. Recent work has indicated that presenilins 1 and 2 may actually be the gamma-secretase enzymes. Presenilin (PS) mutations, which lead to an increase in the production of a longer form of Abeta, are also the most common cause of familial Alzheimer's disease (FAD). Therefore, in an attempt to better characterize the substrate preferences of gamma-secretase, we performed experiments to determine how FAD-linked mutations in PS1 would affect the generation of Abeta peptides from full length precursor substrates that we have previously demonstrated to be proteolytically cleaved at alternative sites and/or by enzymatic activities that are pharmacologically distinct. Presenilin mutations increased the production of Abeta peptides from sites distal to the primary cleavage site ('longer' peptides) and in several cases also decreased production of 'shorter' peptides. These results support a model in which the FAD-linked mutants subtly alter the conformation of the gamma-secretase complex to favor the production of long Abeta.     

Caspase cleaved presenilin-1 is part of active gamma-secretase complexes

gamma-Secretase is a key enzyme involved in the processing of the beta-amyloid precursor protein into amyloid beta-peptides (Abeta). Abeta accumulates and forms plaques in Alzheimer's disease (AD) brains. A progressive neurodegeneration and cognitive decline occurs during the course of the disease, and Abeta is believed to be central for the molecular pathogenesis of AD. Apoptosis has been implicated as one of the mechanisms behind the neuronal cell loss seen in AD. We have studied preservation and activity of the gamma-secretase complex during apoptosis in neuroblastoma cells (SH-SY5Y) exposed to staurosporine (STS). We report that the known components (presenilin, Nicastrin, Aph-1 and Pen-2) interact and form active gamma-secretase complexes in apoptotic cells. In addition, the fragments corresponding to the PS1 N-terminal fragment and the caspase-cleaved PS1 C-terminal fragment (PS1-caspCTF) were found to form active gamma-secretase complexes when co-expressed in presenilin (PS) knockout cells. Interestingly, PS1-caspCTF replaced the normal PS1 C-terminal fragment and was co-immunoprecipitated with the gamma-secretase complex in SH-SY5Y cells exposed to STS. In addition, Abeta was detected in medium from apoptotic HEK APP(swe) cells. Together, the data show that gamma-secretase complexes containing PS1-caspCTF are active, and suggest that this proteolytic activity is also important in dying cells and may affect the progression of AD.     

gamma-Secretase substrate selectivity can be modulated directly via interaction with a nucleotide-binding site

gamma-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin composed of its N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified gamma-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Abeta, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified gamma-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified gamma-secretase preparations; the labeling was blocked by ATP itself and APP-selective gamma-secretase inhibitors. We concluded that a nucleotide-binding site exists within gamma-secretase, and certain compounds that bind to this site can specifically modulate the generation of Abeta while sparing Notch. Drugs targeting the gamma-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease.     

Partial purification and characterization of gamma-secretase from post-mortem human brain

One characteristic feature of Alzheimer's disease is the deposition of amyloid beta-peptide (Abeta) as amyloid plaques within specific regions of the human brain. Abeta is derived from the amyloid beta-peptide precursor protein (beta-APP) by the intramembranous cleavage activity of gamma-secretase. Studies in cells have revealed that gamma-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of gamma-secretase is as yet to be fully elucidated, and no investigations have analyzed gamma-secretase in human brain. To address this we have developed a novel in vitro gamma-secretase activity assay using detergent-solubilized cell membranes and a beta-APP-derived fluorescent probe. We report that human brain-derived gamma-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived gamma-secretase are similar to those described in cells, and proteolysis occurs at the Abeta40- and Abeta42-generating cleavage sites. The ability to isolate gamma-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of beta-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.     

N-glycosylation of human nicastrin is required for interaction with the lectins from the secretory pathway calnexin and ERGIC-53

The gamma-secretase complex, composed of four non-covalently bound transmembrane proteins Presenilin, Nicastrin (NCT), APH-1 and PEN-2, is responsible for the intramembranous cleavage of amyloid precursor protein (APP), Notch and several other type I transmembrane proteins. gamma-Secretase cleavage of APP releases the Abeta peptides, which form the amyloid plaques characteristic of Alzheimer's disease brains, and cleavage of Notch releases an intracellular signalling peptide that is critical for numerous developmental processes. NCT, a type I membrane protein, is the only protein within the complex that is glycosylated. The importance of these glycosylation sites is not fully understood. Here, we have observed that NCT N-linked oligosaccharides mediated specific interactions with the secretory pathway lectins calnexin and ERGIC-53. In order to investigate the role played by N-glycosylation, mutation of each site was performed. All hNCT mutants interacted with calnexin and ERGIC-53, indicating that the association was not mediated by any single N-glycosylation site. Moreover, the interaction with ERGIC-53 still occurred in PS1/2 double knockout cells as detected in immunoprecipitation as well as confocal immunofluorescence microscopy studies, which indicated that NCT interacted with ERGIC-53 prior to its association with the active gamma-secretase complex.     

The gamma-secretase complex: machinery for intramembrane proteolysis

Gamma-secretase is a membrane protease complex that possesses presenilin as a catalytic subunit. Presenilin generates amyloid beta peptides in the brains of Alzheimer's patients and is indispensable to Notch signaling in tissue development and renewal. Recent studies have revealed how presenilin is assembled with its cofactor proteins and acquires the gamma-secretase activity: Aph-1 and nicastrin initially form a subcomplex to bind and stabilize presenilin, and then Pen-2 confers the gamma-secretase activity and facilitates endoproteolysis of presenilin. Understanding the mechanism of gamma-secretase cleavage will help to clarify how intercellular cell signaling through transmembrane proteins is regulated by intramembrane proteolysis, and will hopefully eventually lead to a cure for Alzheimer's disease.     

Gamma-secretase exists on the plasma membrane as an intact complex that accepts substrates and effects intramembrane cleavage

Research on Alzheimer's disease led to the identification of a novel proteolytic mechanism in all metazoans, the presenilin/gamma-secretase complex. This unique intramembrane-cleaving aspartyl protease is required for the normal processing of Notch, Jagged, beta-amyloid precursor protein (APP), E-cadherin, and many other receptor-like proteins. We recently provided indirect evidence of gamma-secretase activity at the cell surface in HeLa cells following inhibition of receptor-mediated endocytosis. Here, we directly identify and isolate gamma-secretase as an intact complex (Presenilin, Nicastrin, Aph-1, and Pen-2) from the plasma membrane, both in overexpressing cell lines and endogenously. Inhibition of its proteolytic activity allowed cell surface gamma-secretase to be captured in association with its plasma membrane-localized APP substrates (C83 and C99). Moreover, non-denaturing isolation of the intact enzyme complex revealed that cell surface gamma-secretase can specifically generate amyloid beta-protein from an APP substrate and similarly cleave a Notch substrate. These data directly establish the proteolytic function of gamma-secretase on the plasma membrane, independent of a hypothesized substrate trafficking role. We conclude that presenilin/gamma-secretase exists as a mature complex at the cell surface, where it interacts with and can cleave its substrates, consistent with an essential function in processing many adhesion molecules and receptors required for cell-cell interaction or intercellular signaling.     

Widespread gamma-secretase activity in the cell, but do we need it at the mitochondria?

gamma-Secretase cleavage of the amyloid precursor protein already subjected to a prior beta-secretase cleavage generates beta-amyloid (Abeta) peptide fragments, which are major constituents of the amyloid plagues found in Alzheimer's disease brain tissues. gamma-Secretase activity and components of the gamma-secretase complex are found in the endoplasmic reticulum-Golgi intermediate compartment, the Golgi, the trans-Golgi network, the plasma membrane, the endosomal-lysosomal system and recently, the mitochondria. Abeta fragments have been shown to be neurotoxic, leading to mitochondrial dysfunction and enhanced apoptotic cell death. However, if Abeta fragments are indeed detrimental to neurons, the widespread presence of enzymatic activity that would result in their generation in the cell appears to make little sense. The presence of a gamma-secretase complex in the mitochondrion, an organelle that is particularly susceptible to Abeta toxicity, is even more puzzling. Emerging evidence suggests that both secreted and intracellular Abeta fragments have endogenous functions. Also, while the fibrillogenic Abeta1-42 is clearly neurotoxic, the more abundant and soluble Abeta1-40 is an antioxidant and could potentially be neuroprotective in several ways. A "physiological" amount of Abeta1-40 production by cellular gamma-secretase activity may be part of the neuron's natural counter against oxidative damage, in addition to endogenous roles in neuronal survival and modulation of synaptic transmission. In any case, whether Abeta is produced locally in the mitochondria and the function for mitochondrial Abeta, if produced, is yet unclear.     

Active gamma-secretase is localized to detergent-resistant membranes in human brain

Several lines of evidence suggest that polymerization of the amyloid beta-peptide (Abeta) into amyloid plaques is a pathogenic event in Alzheimer's disease (AD). Abeta is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by beta-secretase and gamma-secretase, and it has been suggested that these enzymes could be targets for treatment of AD. gamma-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that gamma-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied gamma-secretase in detergent-resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in gamma-secretase components and activity. The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the gamma-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co-immunoprecipitation studies further supported the notion that the gamma-secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in gamma-secretase activity, suggesting that DRMs from fresh rat brain may be useful for gamma-secretase activity studies. Finally, confocal microscopy showed co-localization of gamma-secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active gamma-secretase complex is localized to lipid rafts in human brain.     

Purification and characterization of the human gamma-secretase complex

Gamma-secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a approximately 100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high Abeta40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.     

Active gamma-secretase complexes contain only one of each component

Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.     

Intramembrane proteolysis by gamma-secretase

Gamma-secretase mediates the final proteolytic cleavage, which liberates amyloid beta-peptide (Abeta), the major component of senile plaques in the brains of Alzheimer disease patients. Therefore, gamma-secretase is a prime target for Abeta-lowering therapeutic strategies. gamma-Secretase is a protein complex composed of four different subunits, presenilin (PS), APH-1, nicastrin, and PEN-2, which are most likely present in a 1:1:1:1 stoichiometry. PS harbors the catalytically active site, which is critically required for the aspartyl protease activity of gamma-secretase. Moreover, numerous familial Alzheimer disease-associated mutations within the PSs increase the production of the aggregation-prone and neurotoxic 42-amino acid Abeta. Nicastrin may serve as a substrate receptor, although this has recently been challenged. PEN-2 is required to stabilize PS within the gamma-secretase complex. No particular function has so far been assigned to APH-1. The four components are sufficient and required for gamma-secretase activity. At least six different gamma-secretase complexes exist that are composed of different variants of PS and APH-1. All gamma-secretase complexes can exert pathological Abeta production. Assembly of the gamma-secretase complex occurs within the endoplasmic reticulum, and only fully assembled and functional gamma-secretase complexes are transported to the plasma membrane. Structural analysis by electron microscopy and chemical cross-linking reveals a water-containing cavity, which allows intramembrane proteolysis. Specific and highly sensitive gamma-secretase inhibitors have been developed; however, they interfere with the physiological function of gamma-secretase in Notch signaling and thus cause rather significant side effects in human trials. Modulators of gamma-secretase, which selectively affect the production of the pathological 42-amino acid Abeta, do not inhibit Notch signaling.     

A presenilin-independent aspartyl protease prefers the gamma-42 site cleavage

beta-Amyloid peptides (Abeta40 and Abeta42) are the major constituents of amyloid plaques, which are one of the hallmarks of Alzheimer's disease (AD). The Abeta is derived from sequential cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. gamma-Secretase consists of at least four proteins where presenilins (PS1 and PS2 or PS) are the catalytic subunit involved in the gamma-site cleavage of APP. Secretion of both Abeta40 and Abeta42 is significantly reduced in PS1 knock-out cells and completely abolished in cells deficient for both PS1 and PS2. Consequently, both the PS proteins play essential roles in the production of the secretory of Abeta from cells. Recent studies in primary neurons, however, suggest that PSs are not required for intracellular Abeta42 accumulation; thus the intracellular Abeta42 appears to be generated in a PS-independent manner. Here we present the first biochemical evidence indicating that Abeta, especially Abeta42, can be generated in the absence of PS based on an in vitrogamma-secretase assay employing membranes prepared from PS-deficient Blastocyst-derived (BD) cells. This PS-independent gamma-secretase (PSIG) activity is sensitive to the changes in pH and displays an optimal activity at pH 6.0. Pepstatin A is a potent inhibitor for this proteolytic activity with IC50 of 1.2 nm and 0.4 nm for Abeta40 and Abeta42 generation, respectively. These results indicate that these PS-independent gamma-site cleavages are mediated by an aspartyl protease. More importantly, the PSIG activity displays a distinct preference in mediating the 42-site cleavage over the 40-site cleavage, thereby generating Abeta42 as the predominant product.     

The Nicastrin ectodomain adopts a highly thermostable structure

Nicastrin is a type I transmembrane glycoprotein, which is part of the high molecular weight γ-secretase complex. γ-Secretase is one of the key players associated with the generation of Alzheimer's disease pathology, since it liberates the neurotoxic amyloid β-peptide. Four proteins Nicastrin, anterior pharynx-defective-1 (Aph-1), presenilin enhancer-2 (Pen-2) and Presenilin are essential to form the active γ-secretase complex. Recently it has been shown, that Nicastrin has a key function in stabilizing the mature γ-secretase complex and may also be involved in substrate recognition. So far no structural data for the Nicastrin ectodomain or any other γ-secretase component are available. We therefore used Circular Dichroism (CD) spectroscopy to demonstrate that Nicastrin, similar to its homologues, the Streptomyces griseus aminopeptidase (SGAP) and the transferrin receptor (TfR), adopts a thermostable secondary structure. Furthermore, the Nicastrin ectodomain has an exceptionally high propensity to refold after thermal denaturation. These findings provide evidence to further support the hypothesis that Nicastrin may share evolutionary conserved properties with the aminopeptidase and the transferrin receptor family.     

Pen-2 is sequestered in the endoplasmic reticulum and subjected to ubiquitylation and proteasome-mediated degradation in the absence of presenilin

The gamma-secretase complex catalyzes intramembrane proteolysis of a number of transmembrane proteins, including amyloid precursor protein, Notch, ErbB4, and E-cadherin. gamma-Secretase is known to contain four major protein constituents: presenilin (PS), nicastrin, Aph-1, and Pen-2, all of which are integral membrane proteins. There is increasing evidence that the formation of the complex and the stability of the individual components are tightly controlled in the cell, assuring correct composition of functional complexes. In this report, we investigate the topology, localization, and mechanism for destabilization of Pen-2 in relation to PS function. We show that PS1 regulates the subcellular localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature gamma-secretase complexes reside. PS deficiency also leads to destabilization of Pen-2, which is alleviated by proteasome inhibitors. In keeping with this, we show that Pen-2, which adopts a hairpin structure with the N and C termini facing the luminal space, is ubiquitylated prior to degradation and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our data suggest that failure to become incorporated into the gamma-secretase complex leads to degradation of Pen-2 through the ER-associated degradation-proteasome pathway.     

APH1, PEN2, and Nicastrin increase Abeta levels and gamma-secretase activity

A major component of the amyloid plaque core in Alzheimer's disease (AD) is the 40-42-residue amyloid beta peptide (Abeta). Mutations linked to AD such as those in presenilins 1 (PS1) and 2 (PS2) invariably increase the longer Abeta42 species that forms neurotoxic oligomers. It is believed that PS1/2 constitute the catalytic subunit of the gamma-secretase responsible for the final step in Abeta biogenesis. Recent genetic studies have identified a number of additional genes encoding APH1a, APH1b, PEN2, and Nicastrin proteins, which are part of the gamma-secretase complex with PS1. Further, knockout studies using RNAi showed that these components are essential for gamma-secretase activity. However, the nature of gamma-secretase and how the aforementioned proteins regulate its activity are still incompletely understood. Here we present evidence that unlike PS1, overexpression of these proteins can increase the levels of Abeta, suggesting that these proteins are limiting for gamma-secretase activity. In addition, our studies also suggest that the presenilin partners regulate the relative levels of Abeta40 and Abeta42.     

Notch and presenilin: a proteolytic mechanism emerges.

Presenilins are needed for proteolytic processing of transmembrane proteins of the Notch/Lin-12 family and for cleavage of the amyloid precursor protein. Accumulating evidence now strongly implicates Presenilin as the catalytic core of a multiprotein complex that executes an unusual intramembranous cleavage of its substrates. In the case of amyloid precursor protein, this cleavage contributes to the generation of small, toxic amyloid peptides that trigger the pathological development of Alzheimer's disease. In the Notch/Lin-12 pathway, Presenilin-mediated cleavage of the receptor is a crucial feature of ligand-induced receptor activation and signal transduction. In this pathway, the Presenilins perform a regulated cleavage event that follows additional processing steps during receptor maturation and ligand-induced ectodomain removal.     

The extreme C terminus of presenilin 1 is essential for gamma-secretase complex assembly and activity

The gamma-secretase complex catalyzes the cleavage of the amyloid precursor protein in its transmembrane domain resulting in the formation of the amyloid beta-peptide and the cytoplasmic APP intracellular domain. The active gamma-secretase complex is composed of at least four subunits: presenilin (PS), nicastrin, Aph-1, and Pen-2, where the presence of all components is critically required for gamma-cleavage to occur. The PS proteins are themselves subjected to endoproteolytic cleavage resulting in the generation of an N-terminal and a C-terminal fragment that remain stably associated as a heterodimer. Here we investigated the effects of modifications on the C terminus of PS1 on PS1 endoproteolysis, gamma-secretase complex assembly, and activity in cells devoid of endogenous PS. We report that certain mutations and, in particular, deletions of the PS1 C terminus decrease gamma-secretase activity, PS1 endoproteolysis, and gamma-secretase complex formation. We demonstrate that the N- and C-terminal PS1 fragments can associate with each other in mutants having C-terminal truncations that cause loss of interaction with nicastrin and Aph-1. In addition, we show that the C-terminal fragment of PS1 alone can mediate interaction with nicastrin and Aph-1 in PS null cells expressing only the C-terminal fragment of PS1. Taken together, these data suggest that the PS1 N- and C-terminal fragment intermolecular interactions are independent of an association with nicastrin and Aph-1, and that nicastrin and Aph-1 interact with the C-terminal part of PS1 in the absence of an association with full-length PS1 or the N-terminal fragment.     

Reconstitution of gamma-secretase activity

gamma-Secretase is a membrane protein complex with an unusual aspartyl protease activity that catalyses the regulated intramembranous cleavage of the beta-amyloid precursor protein (APP) to release the Alzheimer's disease (AD)-associated amyloid beta-peptide (Abeta) and the APP intracellular domain (AICD). Here we show the reconstitution of gamma-secretase activity in the yeast Saccharomyces cerevisiae, which lacks endogenous gamma-secretase activity. Reconstituted gamma-secretase activity depends on the presence of four complex components including presenilin (PS), nicastrin (Nct), APH-1 (refs 3-6) and PEN-2 (refs 4, 7), is associated with endoproteolysis of PS, and produces Abeta and AICD in vitro. Thus, the biological activity of gamma-secretase is reconstituted by the co-expression of human PS, Nct, APH-1 and PEN-2 in yeast.