Aph-1 interacts at the cell surface with proteins in the active gamma-secretase complex and membrane-tethered Notch

The activity of the gamma-secretase complex is critical for the processing of a number of transmembrane proteins, including Notch. Functional gamma-secretase activity can be reconstituted from four proteins--presenilin, nicastrin, Pen-2 and Aph-1--but the role of the individual proteins remains unclear. In this report we describe the cellular localization and protein interactions of Aph-1, with particular regard to Notch receptor processing. We found that Aph-1 is present at the cell surface, where it interacts with Pen-2, the mature forms of presenilin and nicastrin, and full-length Notch. Aph-1 also interacts with a truncated form of Notch, which is a direct substrate for gamma-secretase, but not with the Notch intracellular domain. Immunoprecipitation data for Notch and Aph-1 showed that the Notch-containing gamma-secretase complexes most likely form a small subset of the total number of gamma-secretase complexes. In conclusion, these data demonstrate that Aph-1 is present at the cell surface, presumably in active gamma-secretase complexes, and interacts with the Notch receptor, both before and after ligand activation.     

Different cofactor activities in gamma-secretase assembly: evidence for a nicastrin-Aph-1 subcomplex

The gamma-secretase complex is required for intramembrane cleavage of several integral membrane proteins, including the Notch receptor, where it generates an active signaling fragment. Four putative gamma-secretase components have been identified-presenilin (Psn), nicastrin (Nct), Aph-1, and Pen-2. Here, we use a stepwise coexpression approach to investigate the role of each new component in gamma-secretase assembly and activation. Coexpression of all four proteins leads to high level accumulation of mature Psn and increased proteolysis of Notch. Aph-1 and Nct may form a subcomplex that stabilizes the Psn holoprotein at an early step in gamma-secretase assembly. Subcomplex levels of Aph-1 are down-regulated by stepwise addition of Psn, suggesting that Aph-1 might not enter the mature complex. In contrast, Pen-2 accumulates proportionally with Psn, and is associated with Psn endoproteolysis during gamma-secretase assembly. These results demonstrate that Aph-1 and Pen-2 are essential cofactors for Psn, but that they play different roles in gamma-secretase assembly and activation.     

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.     

Assembly of the gamma-secretase complex involves early formation of an intermediate subcomplex of Aph-1 and nicastrin

The gamma-secretase complex is an unusual multimeric protease responsible for the intramembrane cleavage of a variety of type 1 transmembrane proteins, including the beta-amyloid precursor protein and Notch. Genetic and biochemical data have revealed that this protease consists of the presenilin heterodimer, a highly glycosylated form of nicastrin, and the recently identified gene products, Aph-1 and Pen-2. Whereas current evidence supports the notion that presenilin comprises the active site of the protease and that the other three components are members of the active complex required for proteolytic activity, the individual roles of the three co-factors remain unclear. Here, we demonstrate that endogenous Aph-1 interacts with an immature species of nicastrin, forming a stable intermediate early in the assembly of the gamma-secretase complex, prior to the addition of presenilin and Pen-2. Our data suggest 1) that Aph-1 is involved in the early stages of gamma-secretase assembly through the stabilization and perhaps glycosylation of nicastrin and by scaffolding nicastrin to the immature gamma-secretase complex, and 2) that presenilin, and later Pen-2, bind to this intermediate during the formation of the mature protease.     

Aph-1 contributes to the stabilization and trafficking of the gamma-secretase complex through mechanisms involving intermolecular and intramolecular interactions

Gamma-secretase cleaves type I transmembrane proteins, including beta-amyloid precursor protein and Notch, and requires the formation of a protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2 for its activity. Aph-1 is implicated in the stabilization of this complex, although its precise mechanistic role remains unknown. Substitution of the first glycine within the transmembrane GXXXG motif of Aph-1 causes a loss-of-function phenotype in Caenorhabditis elegans. Here, using an untranslated region-targeted RNA interference/rescue strategy in Drosophila Schneider 2 cells, we show that Aph-1 contributes to the assembly of the gamma-secretase complex by multiple mechanisms involving intermolecular and intramolecular interactions depending on or independent of the conserved glycines. Aph-1 binds to nicastrin forming an early subcomplex independent of the conserved glycines within the endoplasmic reticulum. Certain mutations in the conserved GXXXG motif affect the interaction of the Aph-1.nicastrin subcomplex with presenilin that mediates trafficking of the presenilin.Aph-1.nicastrin tripartite complex to the Golgi. The same mutations decrease the stability of Aph-1 polypeptides themselves, possibly by affecting intramolecular associations through the transmembrane domains. Our data suggest that the proper assembly of the Aph-1.nicastrin subcomplex with presenilin is the prerequisite for the trafficking as well as the enzymatic activity of the gamma-secretase complex and that Aph-1 functions as a stabilizing scaffold in the assembly of this complex.     

Detergent-dependent dissociation of active gamma-secretase reveals an interaction between Pen-2 and PS1-NTF and offers a model for subunit organization within the complex

Gamma-secretase is a member of a new class of proteases with an intramembrane catalytic site and cleaves numerous type I membrane proteins, including the amyloid beta-protein precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: a heterodimeric form of presenilin (PS), composed of its N- and C-terminal fragments (PS-NTF and PS-CTF, respectively), a highly glycosylated, mature form of nicastrin (NCT), Aph-1, and Pen-2. However, it is unclear how these components interact physically with each other and assemble into functional complexes. We and others recently found that Aph-1 interacts with a less glycosylated, immature form of nicastrin as an intermediate toward full assembly of gamma-secretase. Here we show that (1) the detergent dodecyl beta-d-maltoside (DDM) mediates the dissociation and inactivation of active gamma-secretase in a concentration-dependent manner, (2) DDM-dependent dissociation of the active gamma-secretase complex generates two major inactive complexes (Pen-2-PS1-NTF and mNCT-Aph-1) and two minor inactive complexes (mNCT-Aph1-PS1-CTF and PS1-NTF-PS1-CTF), and (3) Pen-2 can also associate with the PS holoprotein in complexes devoid of NCT and Aph-1. Taken together, our results demonstrate that Pen-2 interacts with PS-NTF within active gamma-secretase and offer a model for how the components of active gamma-secretase interact physically with each other.     

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.     

Pen-2 is incorporated into the gamma-secretase complex through binding to transmembrane domain 4 of presenilin 1

gamma-Secretase is a multimeric membrane protein complex comprised of presenilin (PS), nicastrin (Nct), Aph-1, and Pen-2. It is a member of an atypical class of aspartic proteases that hydrolyzes peptide bonds within the membrane. During the biosynthetic process of the gamma-secretase complex, Nct and Aph-1 form a heterodimeric intermediate complex and bind to the C-terminal region of PS, serving as a stabilizing scaffold for the complex. Pen-2 is then recruited into this trimeric complex and triggers endoproteolysis of PS, conferring gamma-secretase activity. Although the Pen-2 accumulation depends on PS, the binding partner of Pen-2 within the gamma-secretase complex remains unknown. We reconstituted PS1 in Psen1/Psen2 deficient cells by expressing a series of PS1 mutants in which one of the N-terminal six transmembrane domains (TMDs) was swapped with those of CD4 (a type I transmembrane protein) or CLAC-P (a type II transmembrane protein). We report that the proximal two-thirds of TMD4 of PS1, including the conserved Trp-Asn-Phe sequence, are required for its interaction with Pen-2. Using a chimeric CD4 molecule harboring PS1 TMD4, we further demonstrate that the PS1 TMD4 bears a direct binding motif to Pen-2. Pen-2 may contribute to the activation of the gamma-secretase complex by directly binding to the TMD4 of PS1.     

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.     

Degradation of nicastrin involves both proteasome and lysosome

The glycoprotein nicastrin (NCT) is an essential component of the gamma-secretase complex, a high molecular weight complex which also contains the presenilin proteins, Aph-1 and Pen-2. The gamma-secretase complex is not only involved in APP processing but also in the processing of an increasing number of other type I integral membrane proteins. As the largest subunit of the gamma-secretase complex, NCT plays a crucial role in its activation. Considerable information exists on the distribution, structure and function of NCT; however, little is known of its proteolysis. The present study is aimed at exploring the molecular mechanism of NCT degradation. We found that either proteasomal or lysosomal inhibition can significantly increase the levels of both endogenous and exogenous NCT in various cell lines, and the effect of these inhibitions on NCT was time- and dose-dependent. Immunofluorescent microscopic analysis revealed that NCT accumulates in the ER and Golgi apparatus after proteasomal inhibition, while lysosomal inhibition leads to the accumulation of NCT in the lysosomal apparatus. Co-immunoprecipitation can pull down both NCT and ubiquitin. Taken together, our results demonstrate that NCT degradation involves both the proteasome and the lysosome.     

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.     

Generation and characterization of mutant cell lines defective in gamma-secretase processing of Notch and amyloid precursor protein

Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. We have set up a genetic screen based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. We selected clones deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. We further analyzed two of these clones and identified previously undescribed mutations in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired.     

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.     

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.     

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.     

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.