Gamma-secretase Substrates Research Articles

Inhibition of ??-Secretase as a Therapeutic Intervention for Alzheimer???s Disease

Genetic and experimental evidence points to amyloid-beta (Abeta) peptide as the culprit in Alzheimer's disease pathogenesis. This protein fragment abnormally accumulates in the brain cortex and hippocampus of patients with Alzheimer's disease, and self-aggregates to form toxic oligomers causing neurodegeneration.Abeta is heterogeneous and produced from a precursor protein (amyloid precursor protein [APP]) by two sequential proteolytic cleavages that involve beta- and gamma-secretases. This latter enzyme represents a potentially attractive drug target since it dictates the solubility of the generated Abeta fragment by creating peptides of various lengths, namely Abeta(40) and Abeta(42), the longest being the most aggregating. gamma-Secretase comprises a molecular complex of four integral membrane proteins - presenilin, nicastrin, APH-1 and PEN-2 - and its molecular mechanism remains under extensive scrutiny. The ratio of Abeta(42) over Abeta(40) is increased by familial Alzheimer's disease mutations occurring in the presenilin genes or in APP, near the gamma-secretase cleavage site. Potent gamma-secretase inhibitors have been identified by screening drug libraries or by designing aspartyl protease transition-state analogues based on the APP substrate cleavage site. Most of these compounds are not specific for gamma-secretase cleavage of APP, and equally inhibit the processing of other gamma-secretase substrates, such as Notch and a subset of cell-surface receptors and proteins involved in embryonic development, haematopoiesis, cell adhesion and cell/cell contacts. Therefore, current research aims at finding compounds that show selectivity for APP cleavage, and particularly that inhibit the formation of the aggregating form, Abeta(42). Compounds that target the substrate docking site rather than the enzyme active site are also being investigated as an alternative strategy. The finding that some NSAID analogues preferentially inhibit the formation of Abeta(42) over Abeta(40) and do not affect Notch processing has opened a new therapeutic window. The progress in design of selective inhibitors as well as recent results obtained in animal studies prove that gamma-secretase remains among the best targets for the therapeutic control of amyloid build-up in Alzheimer's disease. The full understanding of gamma-secretase regulation may yet uncover new therapeutic leads.

Analysis of transmembrane domain mutants is consistent with sequential cleavage of Notch by γ‐secretase

gamma-Secretase is a lipid-embedded, intramembrane-cleaving aspartyl protease that cleaves its substrates twice within their transmembrane domains (TMD): once near the cytosolic leaflet (at S3/epsilon) and again in the middle of the TMD (at S4/gamma). To address whether this unusual process occurs in two independent or interdependent steps, we investigated how mutations at the S3/epsilon site in Notch1-based substrates impact proteolysis. We demonstrate that such mutations greatly inhibit not only gamma-secretase-mediated cleavage at S3 but also at S4, independent of their impact on NICD stability. These results, together with our previous observations, suggest that hydrolysis at the center of the Notch transmembrane domain (S4/gamma) is dependent on the S3/epsilon cleavage. Notch (and perhaps all gamma-secretase substrates) may be cleaved by sequential proteolysis starting at S3.

In vitro gamma‐secretase cleavage of the Alzheimer's amyloid precursor protein correlates to a subset of presenilin complexes and is inhibited by zinc

The gamma-secretase complex mediates the final proteolytic event in Alzheimer's disease amyloid-beta biogenesis. This membrane complex of presenilin, anterior pharynx defective, nicastrin, and presenilin enhancer-2 cleaves the C-terminal 99-amino acid fragment of the amyloid precursor protein intramembranously at gamma-sites to form C-terminally heterogeneous amyloid-beta and cleaves at an epsilon-site to release the intracellular domain or epsilon-C-terminal fragment. In this work, two novel in vitro gamma-secretase assays are developed to further explore the biochemical characteristics of gamma-secretase activity. During development of a bacterial expression system for a substrate based on the amyloid precursor protein C-terminal 99-amino acid sequence, fragments similar to amyloid-beta and an epsilon-C-terminal fragment were observed. Upon purification this substrate was used in parallel with a transfected source of substrate to measure gamma-secretase activity from detergent extracted membranes. With these systems, it was determined that recovery of size-fractionated cellular and tissue-derived gamma-secretase activity is dependent upon detergent concentration and that activity correlates to a subset of high molecular mass presenilin complexes. We also show that by changing the solvent environment with dimethyl sulfoxide, detection of epsilon-C-terminal fragments can be elevated. Lastly, we show that zinc causes an increase in the apparent molecular mass of an amyloid precursor protein gamma-secretase substrate and inhibits its cleavage. These studies further refine our knowledge of the complexes and biochemical factors needed for gamma-secretase activity and suggest a mechanism by which zinc dysregulation may contribute to Alzheimer's disease pathogenesis.

The Familial Dementia BRI2 Gene Binds the Alzheimer Gene Amyloid-β Precursor Protein and Inhibits Amyloid-β Production

Alzheimer disease (AD), the most common senile dementia, is characterized by amyloid plaques, vascular amyloid, neurofibrillary tangles, and progressive neurodegeneration. Amyloid is mainly composed by amyloid-beta (A(beta)) peptides, which are derive from processing of the beta-amyloid precursor protein (APP), better named amyloid-beta precursor protein (A(beta)PP), by secretases. The A(beta)PP intracellular domain (AID), which is released together with A(beta), has signaling function, since it modulates apoptosis and transcription. Despite its biological and pathological importance, the mechanisms regulating A(beta)PP processing are poorly understood. As cleavage of other gamma-secretase substrates is regulated by membrane bound proteins, we have postulated the existence of integral membrane proteins that bind A(beta)PP and regulate its processing. Here, we show that BRI2, a type II membrane protein, interacts with A(beta)PP. Interestingly, 17 amino acids corresponding to the NH2-terminal portion of A(beta) are necessary for this interaction. Moreover, BRI2 expression regulates A(beta)PP processing resulting in reduced A(beta) and AID levels. Altogether, these findings characterize the BRI2-A(beta)PP interaction as a regulatory mechanism of A(beta)PP processing that inhibits A(beta) production. Notably, BRI2 mutations cause familial British (FBD) and Danish dementias (FDD) that are clinically and pathologically similar to AD. Finding that BRI2 pathogenic mutations alter the regulatory function of BRI2 on A(beta)PP processing would define dysregulation of A(beta)PP cleavage as a pathogenic mechanism common to AD, FDD, and FBD.

Two domains within the first putative transmembrane domain of presenilin 1 differentially influence presenilinase and γ‐secretase activity

Presenilins (PS) are thought to contain the active site for presenilinase endoproteolysis of PS and gamma-secretase cleavage of substrates. The structural requirements for PS incorporation into the gamma-secretase enzyme complex, complex stability and maturation, and appropriate presenilinase and gamma-secretase activity are poorly understood. We used rescue assays to identify sequences in transmembrane domain one (TM1) of PS1 required to support presenilinase and gamma-secretase activities. Swap mutations identified an N-terminal TM1 domain that is important for gamma-secretase activity only and a C-terminal TM1 domain that is essential for both presenilinase and gamma-secretase activities. Exchange of residues 95-98 of PS1 (sw95-98) completely abolishes both activities while the familial Alzheimer's disease mutation V96F significantly inhibits both activities. Reversion of residue 96 back to valine in the sw95-98 mutant rescues PS function, identifying V96 as the critical residue in this region. The TM1 mutants do not bind to an aspartyl protease transition state analog gamma-secretase inhibitor, indicating a conformational change induced by the mutations that abrogates catalytic activity. TM1 mutant PS1 molecules retain the ability to interact with gamma-secretase substrates and gamma-secretase complex members, although Nicastrin stability is decreased by the presence of these mutants. gamma-Secretase complexes that contain V96F mutant PS1 molecules display a partial loss of function for gamma-secretase that alters the ratio of amyloid-beta peptide species produced, leading to the amyloid-beta peptide aggregation that causes familial Alzheimer's disease.

Low Density Lipoprotein Receptor-related Protein (LRP) Interacts with Presenilin 1 and Is a Competitive Substrate of the Amyloid Precursor Protein (APP) for γ-Secretase

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and the Notch receptor. Recently, the low density receptor-related protein (LRP) has been shown to be cleaved by a gamma-secretase-like activity. We postulated that LRP may interact with PS1 and tested its role as a competitive substrate for gamma-secretase. In this report we show that LRP colocalizes and interacts with endogenous PS1 using coimmunoprecipitation and fluorescence lifetime imaging microscopy. In addition, we found that gamma-secretase active site inhibitors do not disrupt the interaction between LRP and PS1, suggesting that the substrate associates with a gamma-secretase docking site located in close proximity to PS1. This is analogous to APP-gamma-secretase interactions. Finally, we show that LRP competes with APP for gamma-secretase activity. Overexpression of a truncated LRP construct consisting of the C terminus, the transmembrane domain, and a short extracellular portion leads to a reduction in the levels of the Abeta40, Abeta42, and p3 peptides without changing the total level of APP expression. In addition, transfection with the beta-chain of LRP causes an increase in uncleaved APP C-terminal fragments and a concomitant decrease in the signaling effects of the APP intracellular domain. In conclusion, LRP is a PS1 interactor and can compete with APP for gamma-secretase enzymatic activity.

Aph‐1 interacts at the cell surface with proteins in the active γ‐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.

Tumor Necrosis Factor-α, Interleukin-1β, and Interferon-γ Stimulate γ-Secretase-mediated Cleavage of Amyloid Precursor Protein through a JNK-dependent MAPK Pathway

The deposition of the amyloid beta (Abeta) peptide in neuritic plaques plays a critical role in the pathogenesis of Alzheimer's disease (AD). Abeta is generated through the proteolysis of amyloid precursor protein (APP) by the sequential actions of beta- and gamma-secretases. Although recent evidence has unveiled much about the biochemical identity and characteristics of gamma-secretase, the mechanism regulating endogenous gamma-secretase activity remains elusive. To identify possible extracellular signals and associated signaling cascades that could regulate APP proteolysis by gamma-secretase activity, we have developed a cell-based reporter gene assay by stably cotransfecting HEK293 cells with the Gal4-driven luciferase reporter gene and the Gal4/VP16-tagged C-terminal fragment of APP (C99-GV), the immediate substrate of gamma-secretase. The cleavage of C99-GV by gamma-secretase releases the transcription factor that activates luciferase expression, providing a quantitative measurement of gamma-secretase activity. Using this reporter assay, we have demonstrated that interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha can specifically stimulate gamma-secretase activity, concomitant with increased production of Abeta and the intracellular domain of APP (AICD). The gamma-secretase-dependent cleavage of Notch is also enhanced upon the stimulation of these cytokines. The cytokine-enhanced gamma-secretase activity can be suppressed by a potent inhibitor of c-Jun N-terminal kinase (JNK). Furthermore, cells transfected with dominant-positive MEKK1, one of the most potent activators of the JNK cascade, exhibit increased gamma-secretase activity, suggesting that the JNK-dependent mitogen-activated protein kinase pathway could mediate the cytokine-elicited regulation of gamma-secretase. Our studies provide direct evidence that cytokine-elicited signaling cascades control Abeta production by modulating gamma-secretase activity.

Linking Receptor-mediated Endocytosis and Cell Signaling: EVIDENCE FOR REGULATED INTRAMEMBRANE PROTEOLYSIS OF MEGALIN IN PROXIMAL TUBULE

Megalin, a member of the low density lipoprotein receptor gene family, is required for efficient protein absorption in the proximal tubule. Recent studies have shown that the low density lipoprotein receptor-related protein, another member of this gene family, is proteolytically processed by gamma-secretase implying a role for low density lipoprotein receptor-related protein in a Notchlike signaling pathway. This pathway has been shown to involve: 1) metalloprotease-mediated ectodomain shedding and gamma-secretase-mediated intramembrane proteolysis of some receptors. Experiments were performed to determine whether megalin undergoes similar processing. By immunocytochemistry, immunoblotting, and a fluorogenic enzyme assay presenilin-1 (required for gamma-secretase activity) and gamma-secretase activity were found in the brush border of proximal kidney tubules where megalin is localized. Using a fluorogenic peptide containing an amyloid precursor protein gamma-secretase cleavage site and Compound E, a specific gamma-secretase inhibitor, we found high levels of gamma-secretase activity in renal brush border membrane vesicles. Immunoblotting analysis of renal microsomes and opossum kidney proximal tubule (OKP) cells using antibodies directed to the cytosolic domain of megalin showed a 35-40-kDa, membrane-associated, carboxyl-terminal fragment of megalin (MCTF). When cells were incubated with 200 nm phorbol 12-myristate 13-acetate, the appearance of the MCTF increased 2.5-fold and was blocked by metalloprotease inhibitors. When the cells were incubated with gamma-secretase inhibitor Compound E, it caused a 2-fold increase in MCTF. Finally, incubating the cells with 1 microm vitamin D-binding protein resulted in a 25% increase in the appearance of the MCTF. In summary, the MCTF is produced by protein kinase C regulated, metalloprotease-mediated ectodomain shedding and is the substrate for gamma-secretase. We postulate that the enzymatic processing of megalin represents part of a novel ligand-dependent signaling pathway in the proximal tubule that links receptor-mediated endocytosis with cell signaling.

Immature nicastrin stabilizes APH-1 independent of PEN-2 and presenilin: identification of nicastrin mutants that selectively interact with APH-1.

Gamma-secretase is a high molecular mass aspartyl protease complex composed of presenilin (PS1 or PS2), nicastrin (Nct), anterior pharynx-defective-1 (APH-1) and presenilin enhancer-2 (PEN-2). The complex mediates the intramembraneous proteolysis of beta-secretase cleaved beta-amyloid precursor protein (APP) leading to the secretion of the Alzheimer's disease-associated amyloid beta-peptide (Abeta). In order to dissect functionally important domains of Nct required for gamma-secretase complex assembly, maturation, and activity we mutated evolutionary conserved amino acids. The mutant Nct variants were expressed in a cellular background with significantly reduced endogenous Nct. Mutant Nct was functionally investigated by its ability to restore PS, APH-1 and PEN-2 expression as well as by monitoring the accumulation of the APP C-terminal fragments, the immediate substrates of gamma-secretase. We identified three independent mutations within the ectodomain of Nct, which rescued expression of APH-1 but not of PEN-2 or PS and thus failed to restore gamma-secretase activity. Interestingly, these immature Nct variants selectively bound to APH-1, suggesting a stable Nct/APH-1 interaction independent of PS and PEN-2. Consistent with this finding, expression of APH-1 remained largely unaffected in the PS double knock-out and immature Nct co-immunoprecipitated with APH-1 in the absence of PS and PEN-2. Taken together, our findings suggest that immature Nct can stably interact with APH-1 to form a potential scaffold for binding of PS and PEN-2. Moreover, binding of the latter two complex partners critically depends on the integrity of the Nct ectodomain.

Chronic Treatment with the γ-Secretase Inhibitor LY-411,575 Inhibits β-Amyloid Peptide Production and Alters Lymphopoiesis and Intestinal Cell Differentiation

Inhibition of gamma-secretase, one of the enzymes responsible for the cleavage of the amyloid precursor protein (APP) to produce the pathogenic beta-amyloid (Abeta) peptides, is an attractive approach to the treatment of Alzheimer disease. In addition to APP, however, several other gamma-secretase substrates have been identified (e.g. Notch), and altered processing of these substrates by gamma-secretase inhibitors could lead to unintended biological consequences. To study the in vivo consequences of gamma-secretase inhibition, the gamma-secretase inhibitor LY-411,575 was administered to C57BL/6 and TgCRND8 APP transgenic mice for 15 days. Although most tissues were unaffected, doses of LY-411,575 that inhibited Abeta production had marked effects on lymphocyte development and on the intestine. LY-411,575 decreased overall thymic cellularity and impaired intrathymic differentiation at the CD4(-)CD8(-)CD44(+)CD25(+) precursor stage. No effects on peripheral T cell populations were noted following LY-411,575 treatment, but evidence for the altered maturation of peripheral B cells was observed. In the intestine, LY-411,575 treatment increased goblet cell number and drastically altered tissue morphology. These effects of LY-411,575 were not seen in mice that were administered LY-D, a diastereoisomer of LY-411,575, which is a very weak gamma-secretase inhibitor. These studies show that inhibition of gamma-secretase has the expected benefit of reducing Abeta in a murine model of Alzheimer disease but has potentially undesirable biological effects as well, most likely because of the inhibition of Notch processing.

Notch1 Competes with the Amyloid Precursor Protein for γ-Secretase and Down-regulates Presenilin-1 Gene Expression

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and Notch1. Based on the fact that APP and Notch are processed by the same gamma-secretase, we postulated that APP and Notch compete for the enzyme activity. In this report, we examined the interactions between APP, Notch, and PS1 using the direct gamma-secretase substrates, Notch 1 Delta extracellular domain (N1DeltaEC) and APP carboxyl-terminal fragment of 99 amino acids, and measured the effects on amyloid-beta protein production and Notch signaling, respectively. Additionally, we tested the hypothesis that downstream effects on PS1 expression may coexist with the competition phenomenon. We observed significant competition between Notch and APP for gamma-secretase activity; transfection with either of two direct substrates of gamma-secretase led to a reduction in the gamma-cleaved products, Notch intracellular domain or amyloid-beta protein. In addition, however, we found that activation of the Notch signaling pathway, by either N1 Delta EC or Notch intracellular domain, induced down-regulation of PS1 gene expression. This finding suggests that Notch activation directly engages gamma-secretase and subsequently leads to diminished PS1 expression, suggesting a complex set of feedback interactions following Notch activation.

Presenilin-1 is indirectly implicated in Notch1 cleavage.

It has been previously demonstrated that the Notch1 signalling pathway is impaired in presenilin-1 null cells. This observation suggests a role for presenilin-1 in the Notch1 developmental pathway, possibly through physical interaction. Here, we show that presenilin-1 and Notch1 do not interact directly with each other but are associated in the cell. These findings raise the possibility that the gamma-secretase cleavage occurs via a presenilin complex in association with a putative co-factor specific for the molecule that is being cleaved (e.g. Notch1, (beta-amyloid precursor protein, E-cadherin and ErbB-4, all of which are gamma-secretase substrates).

Aβ42-lowering Nonsteroidal Anti-inflammatory Drugs Preserve Intramembrane Cleavage of the Amyloid Precursor Protein (APP) and ErbB-4 Receptor and Signaling through the APP Intracellular Domain

Epidemiological studies indicate that long term use of nonsteroidal anti-inflammatory drugs (NSAIDs) confers protection from Alzheimer's disease, and some NSAIDs were shown to specifically decrease production of the amyloidogenic Abeta42 peptide, most likely by direct modulation of gamma-secretase activity. In contrast to gamma-secretase inhibitors, Abeta42-lowering NSAIDs do not impair S3 cleavage in the NOTCH receptor and release of the NOTCH intracellular domain, a finding with conceptual implications for the development of safer drugs targeting Abeta production through gamma-secretase modulation. Intramembrane cleavage and release of an intracellular signaling domain has recently been demonstrated in a number of additional gamma-secretase substrates. We now show in cell-based assays that intramembrane cleavage of APP and ErbB-4 receptor is not impaired by the Abeta42-lowering NSAIDs, sulindac sulfide and ibuprofen. Generation of the APP intracellular domain (AICD) was further not inhibited in a cell-free assay at concentrations far exceeding those effective in reducing Abeta42 production. Closer inspection of AICD signaling showed that stabilization of the AICD peptide by FE65 and AICD-mediated transcription were also retained at Abeta42-lowering concentrations. These results demonstrate that S3-like/intramembrane cleavage is preserved by Abeta42-lowering NSAIDs in at least three substrates of gamma-secretase APP, ErbB-4, and NOTCH and underline the striking specificity by which these drugs target Abeta42 production.

Localization of presenilin-nicastrin complexes and gamma-secretase activity to the trans-Golgi network.

Abundant biochemical and genetic evidence suggests that presenilins are catalytic components of gamma-secretase, the protease responsible for generating the Alzheimer amyloid beta-protein. However, the differential localization of presenilins to early secretory compartments and gamma-secretase substrates to late secretory compartments and the plasma membrane (the "spatial paradox") argues against this view. We investigated this issue by studying the localization of nicastrin, another putative gamma-secretase component, and its association with presenilin-1 into proteolytically active complexes. Glycosidase digests revealed that nicastrin exists in multiple glycoforms and is terminally sialylated, a modification often associated with the trans-Golgi network. Trafficking of nicastrin to the trans-Golgi network was confirmed by density gradient fractionation and immunofluorescence microscopy. In presenilin-deficient cells, however, nicastrin trafficking and maturation were abnormal, as the protein was restricted to early secretory compartments and failed to be sialylated. Mature sialylated nicastrin in trans-Golgi network fractions was complexed quantitatively with N- and C-terminal fragments of presenilin-1, whereas immature nicastrin present in early secretory compartments was not. Additionally, trans-Golgi network fractions contained the gamma-secretase substrate beta-amyloid precursor protein C83 and were enriched in presenilin-dependent gamma-secretase proteolytic activity. The results resolve the apparent spatial paradox by demonstrating that presenilin-nicastrin complexes and presenilin-dependent gamma-secretase activity are co-localized to a late secretory compartment. The findings provide further evidence that presenilin-containing complexes are the gamma-secretase, and indicate that presenilins also regulate gamma-secretase assembly.

Mammalian APH-1 Interacts with Presenilin and Nicastrin and Is Required for Intramembrane Proteolysis of Amyloid-β Precursor Protein and Notch

Presenilin and nicastrin are essential components of the gamma-secretase complex that is required for the intramembrane proteolysis of an increasing number of membrane proteins including the amyloid-beta precursor protein (APP) and Notch. By using co-immunoprecipitation and nickel affinity pull-down approaches, we now show that mammalian APH-1 (mAPH-1), a conserved multipass membrane protein, physically associates with nicastrin and the heterodimers of the presenilin amino- and carboxyl-terminal fragments in human cell lines and in rat brain. Similar to the loss of presenilin or nicastrin, the inactivation of endogenous mAPH-1 using small interfering RNAs results in the decrease of presenilin levels, accumulation of gamma-secretase substrates (APP carboxyl-terminal fragments), and reduction of gamma-secretase products (amyloid-beta peptides and the intracellular domains of APP and Notch). These data indicate that mAPH-1 is probably a functional component of the gamma-secretase complex required for the intramembrane proteolysis of APP and Notch.

Failure of the interaction between presenilin 1 and the substrate of gamma-secretase to produce Abeta in insect cells.

Aggregates of beta-amyloid peptide (Abeta) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Abeta results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by beta- and gamma-secretase activities. If beta-secretase has recently been identified and termed BACE, the identity of gamma-secretase is still obscure. Studies with knock-out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the gamma-secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous beta- and/or gamma-secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected beta-secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of gamma-secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Abeta. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the gamma-secretase.

Presenilin-dependent γ-Secretase-like Intramembrane Cleavage of ErbB4

An unusual protease gamma-secretase requires functional presenilins and cleaves substrates (e.g. amyloid beta-protein precursor and Notch) with very loose amino acid sequence specificity within the transmembrane region. Here we report that ErbB4, a tyrosine kinase receptor for neuregulins, is a substrate for presenilin-dependent gamma-secretase. Our studies show that constitutive ectodomain shedding of full-length ErbB4 yields the approximately 80-kDa membrane-associated C-terminal fragment (B4-CTF). Subsequent intramembrane cleavage of the B4-CTF was inhibited in the cells devoid of functional presenilins or by treatment of cells with a gamma-secretase inhibitor, leading to enhanced accumulation of B4-CTF. Furthermore, an in vitro gamma-secretase assay demonstrated that the intracellular domain of ErbB4 (B4-ICD) was produced and subsequently released into the soluble fraction in a presenilin-dependent manner. We have also shown that ectopically expressed B4-ICD is localized to the nucleus, suggesting that the presenilin-dependent cleavage of ErbB4 generates the soluble B4-ICD that functions in the nucleus presumably at transcriptional level. Our study indicates that ErbB4 represents a first receptor tyrosine kinase that undergoes intramembrane proteolysis and may mediate a novel signaling function independent of its canonical role as a receptor tyrosine kinase. Our studies also support the idea that presenilins play a generic role in intramembrane cleavage of selected type I membrane proteins.

Gamma-secretase: substrates and inhibitors.

The amyloid beta-protein (Abeta) deposited in Alzheimer's disease (AD), the most common form of dementia in the elderly, is a secreted proteolytic product of the amyloid beta-protein precursor (APP). Generation of Abeta from the APP requires two sequential proteolytic events, beta-secretase cleavage to generate the amino terminus, followed by gamma-secretase cleavage to generate the carboxyl terminus. Because this process is a central event in the pathogenesis of AD, gamma-secretase is believed to be an excellent therapeutic target. Gamma-secretase activity has been demonstrated to be membrane-associated, with the cleavage site primarily determined by the location of the substrate with respect to the membrane. It has also been shown that this unusual proteolytic activity not only occurs for APP, but also for proteins involved in morphogenic processes or cell proliferation and differentiation such as Notch and ErbB4. Thus far, all gamma-secretase substrates are involved in some form of nuclear signaling. These recent findings have important implications for the development of pharmacological interventions that target gamma-secretase.

Murine Notch Homologs (N1–4) Undergo Presenilin-dependent Proteolysis

Oncogenic forms of Notch1, Notch2, and Notch4 appear to mimic signaling intermediates of Notch1 and suggest that the role of proteolysis in Notch signaling has been conserved. Here we demonstrate that extracellularly truncated Notch homologs are substrates for a presenilin-dependent gamma-secretase activity. Despite minimal conservation within the transmembrane domain, the requirement for a specific amino acid (P1' valine) and its position at the cleavage site relative to the cytosolic border of the transmembrane domain are preserved. Cleaved, untethered Notch intracellular domains from each receptor translocate to the nucleus and interact with the transcriptional regulatory protein CSL. All four Notch proteins display presenilin-dependent transactivating potential on a minimal promoter reporter. Thus, this study increases the number of biochemically characterized gamma-secretase substrates from two to five. Despite a high degree of structural homology and the presenilin-dependent activity of truncated Notch proteins, the extent that this reflects functional redundancy is unknown.