The formation of the stabilized HMW complex of mammalian PS, that requires the integrity of the conserved PS C terminus, is essential to the acquisition of gamma-secretase activity, and an aspartate residue within 7th TMD (TMD7) is crucial to the gamma-secretase activity in mammalian PS. To verify the effects of missense mutations in Psn that cause Notch (i.e, loss-of-function) phenotype in Drosophila in vivo, on the metabolism of Psn polypeptides, the two types of amino acid substitutions (i.e., P507L or G516E) were introduced and stably expressed the mutant Psn in N2a NL/N cells. In addition, N2a NL/N cells, stably coexpressing Psn carrying D461A mutation that replaces the highly conserved aspartate residue in the TMD7 with alanine, were established to see if this mutation works as a dominant negative mutant on gamma-cleavage as in mammalian PS. Neither Psn/P507L, Psn/G516E nor Psn/D461A undergo endoproteolysis to give rise to NTF and CTF that normally occurs with wild type Psn. The replacement of endogenous PS1 did not occur in N2a NL/N cells coexpressing Psn/P507L or Psn/G516E. Upon CHX treatment of the N2a cells, the Psn/P507L or Psn/G516E holoproteins were rapidly degraded in a similar manner to wild type Psn holoprotein. In contrast, the overexpression of Psn/D461A results in a complete replacement of endogenous murine PS1 fragments, and a portion of Psn/D461A is stabilized as a holoprotein, as previously described in aspartate mutants of mammalian PS (i.e., PS1/D385A, PS2/D366A). The HMW complex formation of Psn and its derivatives was analyzed. The unstable Psn/P507L or Psn/G516E holoproteins were fractionated exclusively in the LMW range. In contrast, Psn/D461A, which was stabilized but not cleaved, was present as holoproteins broadly within LMW and HMW ranges in a similar manner to that of mammalian PS2/D366A (Takasugi, 2002).
Psn-dependent gamma-secretase activity in Drosophila has been shown to cleave Notch and other transmembrane proteins in vivo. The amino acid sequence of APPL, a Drosophila homolog of ßAPP, is not homologous to that of mammalian ßAPP especially within the TMD, and gamma-cleavage of APPL has not been documented. However, it has been shown that overexpression of the C-terminal 99 amino acid fragment of human ßAPP elicits the cleavage to generate Aß1-40 by a gamma-secretase-like activity in Drosophila SL-2 cells, although Drosophila cells lack ß-secretase activity. To evaluate the gamma-secretase-like activity for proteolytic processing of the TMD sequence of human ßAPP in Drosophila S2 cells, a cDNA encoding SC100, that corresponds to the C-terminal fragment of human ßAPP starting at the 1st residue of Aß preceded by a signal peptide, was transiently transfected and the conditioned media by ELISA was analyzed. Aß secretion was readily detectable in conditioned media of cells expressing SC100; surprisingly, however, percent Aß42 was ~15%, which was in sharp contrast to the robust Aß1-42 overproduction in mouse N2a cells, that is mediated by the same PS species, i.e., wild type Psn. To exclude the possibility that gamma-secretase-like activity in S2 cells is incapable of producing excessive amounts of Aß1-42, a cDNA encoding SC100 was constructed harboring an isoleucine to phenylalanine substitution at residue 716 of ßAPP (SC100/I716F); this substitution has been shown to cause robust increase in Aß1-42 secretion in COS cells. Transfection of SC100/I716F into S2 cells results in a dramatic increase in Aß1-42 secretion and simultaneous decrease in Aß40 secretion, suggesting that the endogenous gamma-secretase-like activity mediated by Psn normally cleaves the TMD sequence of human ßAPP predominantly at Aß40 position, but is capable of cleaving predominantly at position 42 under pathogenic conditions (e.g., ßAPP mutation) in S2 cells. Thus, Psn-dependent gamma-cleavage in S2 cells shows similar characteristics to those in mammalian cells, whereas it may be shifted to position 42 by some unknown mechanism in mouse N2a cells (Takasugi, 2002).
To examine whether Psn plays an essential role in Aß generation by a gamma-secretase-like activity in S2 cells, an S2 cell line was generated stably expressing SC100 (S2-SC100) and the expression of endogenous Psn gene was suppressed by double-stranded RNA (dsRNA)-mediated interference (RNAi). After a 48-h transfection of Psn dsRNA, the expression of Psn polypeptide in the form of fragments was completely and specifically abolished in S2-SC100 cells. After incubation in fresh media for additional 24 h, the cell lysates and conditioned media were analyzed. Immunoblot analysis has revealed an accumulation of SC100 as well as of a ~10-kDa polypeptide comigrating with C83 of mammalian cells. The latter band presumably represents the SC100 derivative cleaved by an alpha-secretase-like activity, that has been reported in Drosophila and SL-2 cells. No Aß secretion was observed in conditioned media, suggesting that the total suppression of the expression of Psn by RNAi results in a complete loss of gamma-secretase activity. Thus, Psn-dependent gamma-secretase activity is required for Aß generation from a human ßAPP derivative (i.e., SC100) in Drosophila S2 cells (Takasugi, 2002).
Amyloid beta-peptide (Abeta) is generated by the consecutive cuts of two membrane-bound proteases. Beta-secretase cuts at the N terminus of the Abeta domain, whereas gamma-secretase mediates the C-terminal cut. Recent evidence suggests that the presenilin (PS) proteins, PS1 and PS2, may be gamma-secretases. Because PSs principally exist as high molecular weight protein complexes, biologically active gamma-secretases likely require other cofactors such as nicastrin (Nct; see Drosophila Nicastrin) for their activities. Preferentially mature Nct forms a stable complex with PSs. Nct levels have been down-regulated by using a highly specific and efficient RNA interference approach. Very similar to a loss of PS function, down-regulation of Nct levels leads to a massive accumulation of the C-terminal fragments of the beta-amyloid precursor protein. In addition, Abeta production is markedly reduced. Strikingly, down-regulation of Nct destabilizes PS and strongly lowers levels of the high molecular weight PS1 complex. Interestingly, absence of the PS1 complex in PS1(-/-) cells is associated with a strong down-regulation of the levels of mature Nct, suggesting that binding to PS is required for trafficking of Nct through the secretory pathway. Based on these findings it is concluded that Nct and PS regulate each other and determine gamma-secretase function via complex formation (Edbauer, 2002).
The gamma-secretase complex catalyzes the final intramembraneous cleavage of the beta-amyloid precursor protein, liberating the neurotoxic amyloid beta-peptide implicated in Alzheimer's disease. Apart from the catalytic subunit presenilin (PS), three additional subunits, nicastrin, APH-1, and PEN-2, have been identified. In mammals, two PS homologues, PS1 and PS2, which are part of distinct gamma-secretase complexes, exist. Likewise, two APH-1 homologues, APH-1a and APH-1b, have been identified. Furthermore, two APH-1a splice forms, APH-1aS and APH-1aL, have been reported. Both APH-1a splice forms and APH-1b are expressed in peripheral and neuronal cells. APH-1aS, APH-1aL, and APH-1b form separate, proteolytically active gamma-secretase complexes containing either one of the two PSs. Deficiency of APH-1a causes a decrease in nicastrin, PS, and PEN-2 levels and an increase in the levels of APH-1b, whereas deficiency of APH-1b did not affect the levels of APH-1a or the other complex components. Consistent with this finding, it was found that deficiency of APH-1a is associated with reduced gamma-secretase activity, whereas deficiency of APH-1b is not. Thus, APH-1b gamma-secretase complexes may fulfill redundant functions. Taken together, these results suggest that, dependent on the tissue expression of the individual subunits, six distinct gamma-secretase complexes composed of the known subunits can exist in human cells (Shirotani, 2004).
Studies conducted in cell culture indicate that the gamma-secretase involved in amyloid beta-formation and Notch signaling is a multisubunit aspartic protease. Little is known, however, of the structure, function, or localization of gamma-secretase in the adult brain, or possible effects of familial Alzheimer's disease (FAD)-causing mutations on the brain protease. Mouse brain contains a complex composed of gamma-secretase subunits presenilin-1 N-terminal fragment, presenilin-1 C-terminal fragment, Nicastrin, Aph-1a and Pen-2. A homozygous FAD-linked Presenilin-1 knock-in mutation does not alter relative subunit levels. Immunocytochemical localization of gamma-secretase subunits revealed overlapping but distinct regional and subcellular distributions. All subunits are expressed throughout the neuraxis predominantly in neurons, and are present in axons. Their distributions and levels of expression are unaffected by mutant presenilin-1. In a presenilin-1/amyloid precursor protein double knock-in mouse, subunits are associated with plaques, but are expressed at similar levels in amyloid-rich and -poor regions. gamma-Secretase subunits are distributed much more extensively than circumscribed amyloid deposits, suggesting the importance of other factors for localized amyloid deposition. These results indicate a widespread neuronal function for gamma-secretase in the adult brain, and suggest the pathogenic mechanism of FAD-linked mutations does not involve alterations in the composition, expression or brain distribution of the protease. The subcellular localization of gamma-secretase subunits is consistent with a nerve terminal source for amyloid aggregates (Siman, 2004).
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, it has been shown that mammalian APH-1 (mAPH-1: see Drosophila anterior pharynx defective 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 (Lee, 2002).
APH-1 and PEN-2 genes modulate the function of nicastrin and the presenilins in Caenorhabditis elegans. Preliminary studies in transfected mammalian cells overexpressing tagged APH-1 proteins suggest that this genetic interaction is mediated by a direct physical interaction. Using the APH-1 protein encoded on human chromosome 1 [APH-1(1)L; also known as APH-1a] as an archetype, it is reported that endogenous forms of APH-1 are predominantly expressed in intracellular membrane compartments, including the endoplasmic reticulum and cis-Golgi. APH-1 proteins directly interact with immature and mature forms of the presenilins and nicastrin within high molecular weight complexes that display gamma- and epsilon-secretase activity. Indeed APH-1 proteins can bind to the nicastrin delta312-369 loss of function mutant, which does not undergo glycosylation maturation and is not trafficking beyond the endoplasmic reticulum. The levels of expression of endogenous APH-1(1)L can be suppressed by overexpression of any other members of the APH-1 family, suggesting that their abundance is coordinately regulated. Finally, although the absence of APH-1 destabilizes the presenilins, in contrast to nicastrin and PEN-2, APH-1 itself is only modestly destabilized in cells lacking functional expression of presenilin 1 or presenilin 2. Taken together, these data suggest that APH-1 proteins, and APH-1(1) in particular, may have a role in the initial assembly and maturation of presenilin.nicastrin complexes (Gu, 2003).
The Alzheimer disease-associated presenilin (PS) proteins apparently provide the active site of gamma-secretase, an unusual intramembrane-cleaving aspartyl protease. PSs principally occur as high molecular weight protein complexes that contain nicastrin (Nct) and additional as yet unidentified components. Recently, PEN-2 has been implicated in gamma-secretase function. PEN-2 is a critical component of PS1/gamma-secretase and PS2/gamma-secretase complexes. Strikingly, in the absence of PS1 and PS1/PS2, PEN-2 levels are strongly reduced. Similarly, PEN-2 levels are reduced upon RNA interference-mediated down-regulation of Nct. In contrast, down-regulation of PEN-2 by RNA interference is associated with reduced PS levels, impairs Nct maturation, and deficient gamma-secretase complex formation. It is concluded that PEN-2 is an integral gamma-secretase complex component and that gamma-secretase complex components are expressed in a coordinated manner (Steiner, 2002).
A combination of genetic factors and early life events is thought to determine the vulnerability of an individual to develop a complex neurodevelopmental disorder like schizophrenia. Pharmacogenetically selected, apomorphine-susceptible Wistar rats (APO-SUS) display a number of behavioral and pathophysiological features reminiscent of such disorders. Microarray analyses reveal in APO-SUS rats, relative to their counterpart APO-UNSUS rats, a reduced expression of Aph-1b, a component of the gamma-secretase enzyme complex that is involved in multiple neural developmental signaling pathways. The reduced expression is due to a duplicon-based genomic rearrangement event resulting in an Aph-1b dosage imbalance. The expression levels of the other gamma-secretase components were not affected. However, gamma-secretase cleavage activity was significantly changed, and the APO-SUS/-UNSUS Aph-1b genotypes segregate with a number of behavioral phenotypes. Thus, a subtle imbalance in the expression of a single, developmentally important protein may be sufficient to cause a complex phenotype (Coolen, 2005).
gamma-Secretase is an intramembrane-cleaving aspartyl protease complex that mediates the final cleavage of beta-amyloid precursor protein to liberate the neurotoxic amyloid-beta peptide implicated in Alzheimer's disease. The four proteins presenilin (PS), nicastrin (NCT), APH-1, and PEN-2 are sufficient to reconstitute gamma-secretase activity in yeast. Although PS seems to contribute the catalytic core of the gamma-secretase complex, no distinct function has been attributed to the other components so far. In Caenorhabditis elegans, mutation of a glycine to an aspartic acid within a conserved GXXXG motif in the fourth transmembrane domain of APH-1 causes a loss of function phenotype. Surprisingly, it was found that the human homologue APH-1a carrying the equivalent mutation G122D is fully active in yeast co-expressing PS1, NCT, and PEN-2. To address this discrepancy, APH-1a was expressed as G122D in HEK293 cells. Overexpressed APH-1a G122D os not incorporated into the gamma-secretase complex. Separate overexpression of PS1, NCT, or PEN-2 together with APH-1a G122D allows the formation of heterodimers lacking the other endogenous components. Only the combined overexpression of PS1 and NCT together with APH-1a G122D facilitates the formation of a fully active gamma-secretase complex. Under these conditions, APH-1a G122D supports the production of normal amounts of Abeta. It is concluded that cooperative effects may stabilize a trimeric complex of APH-1a G122D together with PS1 and NCT. Upon successful complex assembly, the GXXXG motif becomes dispensable for gamma-secretase activity (Edbauer, 2005).
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. PS1 was reconstituted 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). The proximal two-thirds of TMD4 of PS1, including the conserved Trp-Asn-Phe sequence, is required for its interaction with Pen-2. Using a chimeric CD4 molecule harboring PS1 TMD4, it was further demonstrated 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 (Watanabe, 2005).
gamma-Secretase is a membrane protein complex that cleaves the beta-amyloid precursor protein (APP) within the transmembrane region, after prior processing by beta-secretase, producing amyloid beta-peptides Abeta(40) and Abeta(42). Errant production of Abeta-peptides that substantially increases Abeta(42) production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1, which contains the proteolytic active site, and three other membrane proteins [nicastrin, anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 (PEN-2)] are required to form the core of the active gamma-secretase complex. This study reports the purification of the native gamma-secretase complexes from HeLa cell membranes and the identification of an additional gamma-secretase complex subunit, CD147 (see Drosophila Basigin), a transmembrane glycoprotein with two Ig-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through coimmunoprecipitation studies of the purified protein from HeLa cells and of solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of Abeta peptides without changing the expression level of the other gamma-secretase components or APP substrates whereas CD147 overexpression has no statistically significant effect on Abeta-peptide production, other gamma-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the gamma-secretase complex down-modulates the production of Abeta-peptides (Zhou, 2005; full text of article).
Macromolecular complexes containing presenilins (PS1 and PS2), nicastrin, anterior pharynx defective phenotype 1 (APH-1), and PS enhancer 2 (PEN-2) mediate the intramembranous, gamma-secretase cleavage of beta-amyloid precursor protein (APP), Notch, and a variety of type 1 membrane proteins. PEN-2 is critical for promoting endoproteolysis of PS1 and the proximal two-thirds of transmembrane domain (TMD) 1 of PEN-2 is required for binding with PS1. This study sought to identify the structural domains of PS1 that are necessary for binding with PEN-2. To address this issue, a series of constructs was generated encoding PS1 mutants harboring deletions or replacements of specific TMDs of PS1-NTF, and the effects of encoded molecules were examined on interactions with PEN-2, stabilization and endoproteolysis of PS1, and gamma-secretase activity. PS1 TMDs 1 and 2 and the intervening hydrophilic loop are dispensable for binding to PEN-2. Furthermore, analysis of chimeric PS1 molecules that harbor replacements of each TMD with corresponding transmembrane segments from the sterol regulatory element-binding protein cleavage activating protein (SCAP) revealed that the PS1-SCAP TMD4 mutant failed to coimmunoprecipitate endogenous PEN-2, strongly suggesting that the fourth TMD of PS1 is required for interaction with PEN-2. Further mutational analyses revealed that the "NF" sequence within the TMD4 of PS1 is the minimal motif that is required for binding with PEN-2, promoting PS1 endoproteolysis and gamma-secretase activity (Kim, 2005).
sel-12 (sel means suppressor/enhancer of lin-12) is a transmembrane protein that facilitates Notch signaling in C. elegans. It is related to Presenilin-1 (S182), a mammalian gene that when mutated causes aggressive, early-onset Alzheimer's via an unknown mechanism. In C. elegans, Notch signaling is involved in the anchor cell/ventral uterine precursor cell (AC/VU) decision and vulval precursor cell (VPC) specification during gonadogenesis. The AC/VU decision involves an interaction between two initially equivalent cells of the somatic gonad. When lin-12, which codes for a Notch family member, is eliminated, both precursor cells become ACs. When lin-12 is constitutively activated, both precursor cells become VUs. sel-12 was isolated as a suppressor of a lin-12 gain of function mutation. That is, sel-12 mutation acts to reduce lin-12 signaling. Reducing sel-12 activity reduces lin-12 activity in lateral signaling that specifies the secondary fate of VPCs. Cell ablation experiments show that sel-12 functions within a VPC to lower lin-12 activity. The predicted SEL-12 protein contains multiple potential transmembrane domains, consistent with its function as a receptor, ligand, channel or membrane structural protein. SEL-12 might be directly involved in lin-12-mediated reception, functioning for example as a co-receptor or as a downstream effector that activatesupon LIN-12 activation. Alternatively, sel-12 may be involved in a more general cellular process, such as receptor localization or recycling, and hence influence lin-12 activity indirectly (Levitan, 1995).
The data presented in this paper suggest that the effect of SEL-12/presenilin on LIN-12/Notch is analogous to its effect on APP. LIN-12/Notch proteins are transmembrane proteins with hallmark motifs: epidermal growth factor-like; LIN-12/Notch repeat, and cdc10/SWI6 (ankyrin) motifs. Like APP, LIN-12/Notch proteins must be correctly sorted and transported to the cell surface, and undergo proteolytic cleavage events. There appears to be at least one constitutive proteolytic cleavage event that occurs in the extracellular domain during the transport to the plasma membrane; the cleaved form produced by this constitutive cleavage event may be the major species present at the cell surface. In addition, binding of ligand appears to induce a cleavage event in or near the transmembrane domain; this apparent cleavage event enables the intracellular domain to translocate to the nucleus, where it participates directly in regulating downstream gene expression. It is conceivable that SEL-12/presenilin is involved in promoting one or more of these cleavage events, either by activating protease(s) or promoting trafficking of either LIN-12 or proteases to an appropriate compartment. The strong accumulation of SEL-12::GFP in the ER/Golgi is consistent with a role for SEL-12 in a constitutive cleavage event involved in maturation of LIN-12/Notch proteins. The fact that less LIN-12::GFP was observed at the cell surface in a sel-12 mutant background could be explained in the context of this model by proposing that abnormal processing of LIN-12 leads to its failure to be transported to the plasma membrane or to its degradation. The putative ligand-dependent cleavage of activated LIN-12 might occur at the plasma membrane or in internalized vesicles. The failure to observe SEL-12::GFP in the plasma membranes of the VPCs does not preclude a role for SEL-12 in ligand-dependent cleavage. It is possible that SEL-12::GFP is present at low abundance in the plasma membrane; the ligand-induced event appears to affect a very small proportion of receptor molecules, suggesting that the agent that promotes the cleavage may not be very abundant. Although the issue of the biochemical mechanism of presenilin function is not resolved in any system, the parallels between APP and LIN-12/Notch trafficking and processing suggest that a common mechanism is involved. An important challenge for the future will be to identify the primary effect of SEL-12/presenilin on APP and LIN-12/Notch, since proteolytic processing, intracellular trafficking and degradation are intimately linked, and altering one process can affect another (Levitan, 1998).
Mutant presenilins have been found to cause Alzheimer disease. This paper describes the identification and characterization of HOP-1, a Caenorhabditis elegans presenilin that displays much more lower sequence identity with human presenilins than does the other C. elegans presenilin, SEL-12. Despite considerable divergence, HOP-1 appears to be a bona fide presenilin, because HOP-1 can rescue the egg-laying defect caused by mutations in sel-12 when hop-1 is expressed under the control of sel-12 regulatory sequences. HOP-1 also has the essential topological characteristics of the other presenilins. Reducing hop-1 activity in a sel-12 mutant background causes synthetic lethality and terminal phenotypes associated with reducing the function of the C. elegans lin-12 and glp-1 genes. These observations suggest that hop-1 is functionally redundant with sel-12 and underscore the intimate connection between presenilin activity and LIN-12/Notch activity inferred from genetic studies in C. elegans and mammals (X. Li, 1997).
Mutations in the human presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease. Studies in Caenorhabditis elegans and in mice indicate that one function of presenilin genes is to facilitate Notch-pathway signaling. Notably, mutations in the C. elegans presenilin gene sel-12 reduce signaling through an activated version of the Notch receptor LIN-12. To investigate the function of a second C. elegans presenilin gene hop-1 and to examine possible genetic interactions between hop-1 and sel-12, a reverse genetic strategy was used to isolate deletion alleles of both loci. Animals bearing both hop-1 and sel-12 deletions display new phenotypes not observed in animals bearing either single deletion. These new phenotypes (germ-line proliferation defects, maternal-effect embryonic lethality, and somatic gonad defects) resemble those resulting from a reduction in signaling through the C. elegans Notch receptors GLP-1 and LIN-12. Thus SEL-12 and HOP-1 appear to function redundantly in promoting Notch-pathway signaling. Phenotypic analyses of hop-1 and sel-12 single and double mutant animals suggest that sel-12 provides more presenilin function than does hop-1. There is as yet no evidence that Notch-pathway signaling is involved in the pathophysiology of Alzheimer's disease (AD). Thus, the relationship between the roles of presenilins in proteolytic processing of APP and in facilitating Notch receptor function remains unclear. Intriguingly, recent evidence suggests that multiple proteolytic processing events are required for intracellular trafficking and signal transduction of the Notch receptor: two cleavage events are proposed to occur in the extracellular domain and a third proposed cleavage occurs within or just carboxyl-terminal to the transmembrane region. The apparent similarities between the processing of APP and Notch, particularly the prospect that both are cleaved within the transmembrane domain, raise the possibility that presenilins affect proteolytic processing of APP and Notch in analogous ways. Presenilins might regulate proteolytic processing directly or might do so indirectly, for example, by promoting normal intracellular trafficking of APP or Notch. In support of a role for presenilins in the processing or trafficking of Notch, LIN-12::GFP levels at the plasma membrane are seen to be reduced in a sel-12 mutant background. An understanding of how presenilins affect Notch-receptor activity may be relevant to an understanding of the way in which presenilins affect APP cleavage and to the identification of targets for preventing the pathophysiological effects of presenilin dysfunction in AD (Westlund, 1999).
aph-2 (Drosophila homolog: CG7012) encodes a novel extracellular protein required for GLP-1-mediated signaling (Goutte, 2000). Aph-2, termed Nicastrin in this study (see Drosophila nicastrin), is a transmembrane glycoprotein that forms high molecular weight complexes with presenilin 1 and presenilin 2. Suppression of nicastrin expression in C. elegans embryos induces a subset of notch/glp-1 phenotypes similar to those induced by simultaneous null mutations in both presenilin homologs of C. elegans (sel-12 and hop-1). Nicastrin also binds carboxy-terminal derivatives of beta-amyloid precursor protein (betaAPP), and modulates the production of the amyloid beta-peptide (Abeta) from these derivatives. Missense mutations in a conserved hydrophilic domain of nicastrin increase Abeta42 and Abeta40 peptide secretion. Deletions in this domain inhibit Abeta production. Nicastrin and presenilins are therefore likely to be functional components of a multimeric complex necessary for the intramembranous proteolysis of proteins such as Notch/GLP-1 and betaAPP (Yu, 2000).
In the absence of homology to other proteins, sequence databases were screened for orthologous genes in other species. A full-length C. elegans nicastrin ortholog was found in public databases (accession no. Q23316; identity = 22%; similarity = 41%). Full-length murine and Drosophila nicastrin orthologs from appropriate cDNA libraries were cloned and sequenced using partial cDNA sequences from these databases as start points (mouse nicastrin accession no. AF24069, identity = 89%, similarity = 93%; D. melanogaster nicastrin accession no. AF240470, identity = 30%, similarity = 48%). The four animal nicastrins have similar predicted topologies and have three domains with significant sequence conservation near residues 306-360, 419-458, and 625-662 of human nicastrin. Within the first conserved domain, all four proteins contained the motif DYIGS (residues 336-340), which is also partially conserved in an Arabidopsis protein. All four animal nicastrins also contain four cysteines spaced at 16 to 17-residue intervals in the N terminus (Cys 195, Cys 213, Cys 230 and Cys 248) (Yu, 2000).
To explore whether nicastrin, like the presenilins, might have a role in Notch signaling in vivo, RNA interference (RNAi) was used in C. elegans. Wild-type worms injected with C. elegans nicastrin double-stranded (ds) RNA produce dead embryos, many of which lack an anterior pharynx. This phenotype is highly reproducible and specific. Except for embryonic lethality, none of the other phenotypes associated with a lack of C. elegans presenilin (sel-12) activity were observed. However, this phenotype is identical to that induced when the activity of genes in the notch/glp-1 pathway (glp-1, aph-1 or aph-2) are reduced, or when the activities of both C. elegans presenilin homologs (sel-12 and hop-1) are reduced simultaneously. Thus nicastrin contributes to some aspects of notch/glp-1 signaling in C. elegans embryos (Yu, 2000).
Nicastrin is a multi-pass transmembrane protein that has recently been identified as a member of high-molecular weight complexes containing presenilin. The C. elegans homolog of nicastrin, aph-2, is required for GLP-1/Notch signaling in the early embryo. In addition to the maternal-effect embryonic lethal phenotype, aph-2 mutant animals also display an egg-laying defect. This latter defect is related to the SEL-12/presenilin egg-laying defect. aph-2 and sel-12 genetically interact and cooperate to regulate LIN-12/Notch signaling in the development of the somatic gonad. In addition, aph-2 and lin-12/Notch genetically interact. A new role for aph-2 in facilitating lin-12 signaling in the somatic gonad is illustrated, thus providing evidence that APH-2 is involved in both GLP-1/Notch- and LIN-12/Notch-mediated signaling events. Nicastrin can partially substitute for aph-2, suggesting a conservation of function between these proteins (Levitan, 2001).
Signaling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of gamma-secretase-mediated cleavage of beta-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimer's disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by gamma-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologs in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signaling pathway. In mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signaling pathway that is affected by PS1. Moreover, several gamma-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of gamma-secretase for the treatment of Alzheimer's disease may risk toxicity caused by reduced Notch signaling (De Strooper, 1999).
Proteolytic release of the Notch-1 intracellular domain (NICD), an essential step in the activation of Notch signaling, is markedly reduced in presenilin-1 (PS1)-deficient cells and is restored by PS1 expression. Nuclear translocation of the NICD is also markedly reduced in PS1-deficient cells, resulting in reduced transcriptional activation. Mutations in PS1 that are associated with familial Alzheimer's disease impair the ability of PS1 to induce proteolytic release of the NICD and nuclear translocation of the cleaved protein. These results suggest that PS1 plays a central role in the proteolytic activation of the Notch-1-signaling pathway and that this function is impaired by pathogenic PS1 mutations. Thus, dysregulation of proteolytic function may underlie the mechanism by which presenilin mutations cause Alzheimer's disease (Song, 1999).
Genetic analyses in Caenorhabditis elegans demonstrate that sel-12 and hop-1, homologs of the Alzheimer's disease-associated presenilin genes, modify signaling through LIN-12 and GLP-1, homologs of the Notch cell surface receptor. To gain insight into the biochemical basis of this genetic interaction, the possibility that presenilin-1 (PS1) physically associates with the Notch1 receptor in mammalian cells was investigated. Notch1 and PS1 coimmunoprecipitate from transiently transfected human embryonic kidney 293 cell lysates in a detergent-sensitive manner, consistent with a noncovalent physical association between the two proteins. The interaction predominantly occurs early in the secretory pathway prior to Notch cleavage in the Golgi, because PS1 immunoprecipitation preferentially recovers the full-length Notch1 precursor. When PS1 is immunoprecipitated from 293 cells that have been metabolically labeled with [35S]methionine and [35S]cysteine, Notch1 is the primary protein detected in PS1 immunoprecipitates, suggesting that this interaction is specific. Furthermore, endogenous Notch and presenilin coimmunoprecipitate from cultured Drosophila cells, indicating that physical interaction can occur at physiological expression levels. These results suggest that the genetic relationship between presenilins and the Notch signaling pathway derives from a direct physical association between these proteins in the secretory pathway (Ray, 1999a).
Mutations in PS genes cause autosomal dominant Alzheimer disease, with age of onset frequently in the 40s. Notch is known as a developmental protein that plays an important role in lateral inhibition and specifying cell fate decisions in proliferating immature cells, and is not known to be present in adult neurons. It was reasoned that, if Notch1/PS-1 interaction is relevant in Alzheimer disease, Notch1 would also need to be expressed in neurons in adult brain and colocalized with PS-1. Notch1, Notch2, and a Notch ligand, Jagged1, are all expressed in adult brain in mouse and in human, with strongest expression in the hippocampal formation and Purkinje cells of the cerebellum. Double immunofluorescent staining demonstrates neuronal colocalization of Notch1 with PS-1. Moreover, Notch1 expression in sporadic Alzheimer disease hippocampus is elevated more than 2-fold in comparison to that in control human hippocampus by both immunohistochemistry and Western blot analysis (p < 0.007). These results support the hypothesis that Notch1 continues to play a role in terminally differentiated neurons, and that Notch1/PS-1 interactions may occur in adult mammalian brain. The alteration in Notch1 expression in sporadic Alzheimer disease raises the possibility that disruption of Notch1/PS-1 functional interactions may occur in Alzheimer disease (Berezovska, 1998).
The normal functional neurobiology of the Alzheimer's disease (AD) related gene presenilin 1 (PS1) is unknown. One clue comes from a genetic screen of Caenorhabditis elegans, which reveals that the presenilin homolog sel-12 facilitates lin-12 function. The mammalian homolog of lin-12, Notch1, is a transmembrane receptor that plays an important role in cell fate decisions during development, including neurogenesis, but does not have a known function in fully differentiated cells. To better understand the potential role of Notch1 in mammalian postmitotic neurons and to test the hypothesis that Notch and PS 1 interact, the effect of Notch1 transfection on neurite outgrowth in primary cultures of hippocampal/cortical neurons was studied. Notch1 inhibits neurite extension, and thus has a function in postmitotic mature neurons in the mammalian CNS. Furthermore, evidence is presented demonstrating that there is a functional interaction between PS1 and Notch1 in mammalian neurons, analogous to the sel-12/lin-12 interaction in vulval development in C. elegans. The inhibitory effect of Notch1 on neurite outgrowth is markedly attenuated in neurons from PS1 knockout mice, and enhanced in neurons from transgenic mice overexpressing wild type PS1, but not mutant PS1. These data suggest that PS1 facilitates Notch1 function in mammalian neurons, and support the hypothesis that a functional interaction exists between PS1 and Notch1 in postmitotic mammalian neurons (Berezovska, 1999).
Presenilin-1 (PS1), a polytopic membrane protein primarily localized to the endoplasmic reticulum, is required for efficient proteolysis of both Notch and beta-amyloid precursor protein (APP) within their trans-membrane domains. The activity that cleaves APP (called gamma-secretase) has properties of an aspartyl protease; mutation of either of the two aspartate residues located in adjacent transmembrane domains of PS1 inhibits gamma-secretase processing of APP. These aspartates are required for Notch processing, since mutation of these residues prevents PS1 from inducing the gamma-secretase-like proteolysis of a Notch1 derivative. Thus PS1 might function in Notch cleavage as an aspartyl protease or di-aspartyl protease cofactor. However, the ER localization of PS1 is inconsistent with that hypothesis, since Notch cleavage occurs near the cell surface. Using pulse-chase and biotinylation assays, evidence is provided that PS1 binds Notch in the ER/Golgi and is then co-transported to the plasma membrane as a complex. PS1 aspartate mutants are indistinguishable from wild-type PS1 in their ability to bind Notch or traffic with it to the cell surface, and do not alter the secretion of Notch. Thus, PS1 appears to function specifically in Notch proteolysis near the plasma membrane as an aspartyl protease or cofactor (Ray, 1999b).
Mouse Notch1, which plays an important role in cell fate determination in development, is proteolytically processed within its transmembrane domain by unidentified gamma-secretase-like activity that depends on presenilin. To study this proteolytic event, a cell-free Notch cleavage assay system was established using the membrane fraction of fibroblast transfectants of various Notch constructs with deletion of the extracellular portion (NotchDeltaE). The cytoplasmic portion of Notch1DeltaE is released from the membrane upon incubation at 37°C; this is inhibited by the specific gamma-secretase inhibitor, MW167, or by overexpression of dominant negative presenilin1. Likewise, other members of mouse Notch family are proteolytically cleaved in a presenilin-dependent, MW167-sensitive manner in vivo as well as in the cell-free Notch DeltaE cleavage assay system. All four members of the mouse Notch family migrate to the nucleus and activate the transcription from the promoter carrying the RBP-J consensus sequences after they are released from the membrane. These results demonstrate the conserved biochemical mechanism of signal transduction among mammalian Notch family members (Mizutani, 2001).
Following ectodomain shedding, Notch-1 undergoes presenilin (PS)-dependent constitutive intramembranous endoproteolysis at site-3. This cleavage is similar to the PS-dependent g-secretase cleavage of the ß-amyloid precursor protein (ßAPP). However, topological differences in cleavage resulting in amyloid ß-peptide (Aß) or the Notch-1 intracellular domain (NICD) indicate independent mechanisms of proteolytic cleavage. The secretion of an N-terminal Notch-1 Aß-like fragment (Nß) is reported in this study. Analysis of Nß by MALDI-TOF MS revealed that Nß is cleaved at a novel site (site-4, S4) near the middle of the transmembrane domain. Like the corresponding cleavage of ßAPP at position 40 and 42 of the Aß domain, S4 cleavage is PS dependent. The precision of this cleavage is affected by familial Alzheimer’s disease-associated PS1 mutations similar to the pathological endoproteolysis of ßAPP. Considering these similarities between intramembranous processing of Notch and ßAPP, it is concluded that these proteins are cleaved by a common mechanism utilizing the same protease, i.e. PS/g-secretase (Okochi, 2002).
Notch receptors undergo a cascade of endoproteolytic cleavages required for Notch signaling. Upon binding of membrane-anchored ligands from the DSL (Delta/Serrate/Lag-2) family, Notch receptors undergo consecutive cleavages at site-2 (S2) and site-3 (S3). Cleavage of mouse Notch-1 at S2 occurs in its ectodomain by TACE [tumor necrosis factor-a (TNF-a-converting enzyme], a member of the ADAM (a disintegrin and metalloprotease domain) family ~12 amino acids distant from the TM. This 'ectodomain shedding' event results in the generation of NEXT (Notch extracellular truncation), that is cleaved subsequently at S3 within the TM close to the cytoplasmic border. Cleavage of Notch at S3 liberates NICD (Notch intracellular domain) that translocates to the nucleus, where it is involved in target gene transcription. S3 cleavage strictly depends on the biological activity of the presenilin (PS) proteins, which may contribute the catalytic site of g-secretase, an unusual intramembrane-cleaving aspartyl protease complex (Okochi, 2002 and references therein).
Beside the Notch-1-4 receptors, several other type I TM proteins have been identified as substrates for PS-dependent endoproteolysis, including the Alzheimer's disease (AD)-associated ß-amyloid protein precursor (ßAPP), ErbB-4, E-cadherin and LRP. These proteins undergo ‘ectodomain shedding’ in their large extracellular domains, prior to the consecutive PS-dependent cleavage within the TM. In the case of ßAPP, these cleavages are mediated by a-secretase and ß-secretase. Cleavage of ßAPP by a- and ß-secretase (BACE) results in the generation of the respective ßAPP C-terminal fragments (CTFs), CTFa and CTFß, which are the direct substrates for g-secretase cleavage. Cleavage of CTFß and CTFa by g-secretase occurs in the middle of the TM and leads to the liberation of Aß and p3 peptides), respectively. Aß is deposited in the brain of AD patients in 'senile plaques', an invariant pathological hallmark of AD. Recently, the elusive C-terminal cleavage product of g-secretase, AICD (ßAPP intracellular domain), has been identified and characterized. Surprisingly, AICD results from PS-dependent g-secretase cleavage of ßAPP-CTFs predominantly after Leu49 (Aß numbering). This cleavage is almost identical to the S3 cleavage of Notch-1 and does not occur after Val40 and Ala42 (Aß numbering) as predicted. Thus, g-secretase cleaves the ßAPP TM at several sites: one in the middle after position 40 (g40) and 42 (g42) (with major g40 and minor g42 cleavage) and one close to the cytoplasmic border after position 49 (g49) of the Aß domain. Interestingly, AICD may translocate to the nucleus where it could have a role in transcriptional regulation similar to NICD (Okochi, 2002 and references therein).
Because of these striking similarities between Notch and ßAPP endoproteolysis, it was hypothesized that an Aß/p3-like species (called Notch ß-peptide, Nß) derived from NEXT intramembranous proteolysis may be secreted into the extracellular space. This study reports the identification and characterization of secreted Nß peptides derived from endoproteolysis of NEXT derivatives. Sequence analysis revealed that Nß is derived from endoproteolytic cleavage near the middle of the Notch-1 TM at site-4 (S4), which is 12 amino acid residues upstream of S3. Like S3 cleavage, S4 cleavage occurs in a PS- and g-secretase-dependent manner. Strikingly, familial AD (FAD)-associated PS mutants known to cause the increased production of C-terminally elongated pathogenic Aß42 also affect the generation of C-terminally elongated Nß variants, supporting a direct role for PS in the proteolytic cleavage of Notch-1 and ßAPP (Okochi, 2002).
The role of Notch signaling in general and presenilin in particular during mouse somitogenesis was analyzed. Cyclical production of activated Notch (NICD) was visualized and it was established that somitogenesis requires less NICD than any other tissue in early mouse embryos. Indeed, formation of cervical somites proceeds in Notch1; Notch2-deficient embryos. This is in contrast to mice lacking all presenilin alleles, that have no somites. Since Nicastrin-, Pen-2-, and APH-1a-deficient embryos have anterior somites without γ-secretase, presenilin may have a γ-secretase-independent role in somitogenesis. Embryos triple homozygous for both presenilin null alleles and a Notch allele that is a poor substrate for presenilin (N1V→G) experience fortuitous cleavage of N1V→G by another protease. This restores NICD, anterior segmentation, and bilateral symmetry but does not rescue rostral/caudal identities. These data clarify multiple roles for Notch signaling during segmentation and suggest that the earliest stages of somitogenesis are regulated by both Notch-dependent and Notch-independent functions of presenilin (Hupper, 2005).
Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. A modification, monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. The lysine residue targeted by the monoubiquitination event has been identified, and its importance for activation of Notch receptor by its ligand, Delta-like 1, has been confirmed. A new model is proposed where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and its relevance for other gamma-secretase substrates is discussed (Gupta-Rossi, 2004).
The ubiquitination pathway involves a multiprotein cascade in which the substrate specificity is determined by the E3 component. Multi-ubiquitin chains at least four subunits long are required for efficient recognition and degradation of ubiquitinated proteins by the proteasome, but ubiquitin has more recently been shown to endorse new functions that do not always involve the proteasome (Gupta-Rossi, 2004).
The results show that a monoubiquitination event takes place on the DeltaE molecule, a constitutively active form of the Notch receptor that mimics the intermediate TACE-processing product generated after ligand binding. This modification is a prerequisite for gamma-secretase cleavage and targets one of the subunits of a dimeric membrane-anchored form of Notch DeltaE. The major site of monoubiquitination has been localized to a juxtamembrane, conserved lysine residue K1749 in mNotch1. Access to the monoubiquitinated form DeltaEu was gained by coimmunoprecipitation with endogenous PS1 when gamma-secretase activity was inhibited by a specific drug. Thus, DeltaEu is a labile intermediate appearing before gamma-secretase cleavage. This form could also be detected after coexpression of PS1 or DeltaC4, a PS2-derived construct. These molecules are probably not included in PS-containing high molecular weight complexes; neither are gamma-secretase components when transiently overexpressed. The existence of DeltaEu was varified by extracting and stabilizing it out of the active complexes. It is proposed that this ubiquitination step is required in the context of the full-length receptor activated by ligand binding. Although the modified intermediate species derived from full-length Notch could not be directly accessed, mutating the crucial lysine residue impaired Dll1-mediated Notch signaling, in accordance with the DeltaE results. It remains to be determined which E3 ubiquitin ligase is involved in this modification. Various proteins carrying such an activity have been associated with the Notch cascade and are candidates to be tested, e.g., Deltex, Suppressor of Deltex, and Cbl. Experiments are in progress to answer this question (Gupta-Rossi, 2004).
The results show that endocytosis of Notch DeltaE and of ligand-activated full-length Notch are necessary for gamma-secretase cleavage. The involvement of a clathrin-dependent endocytosis event for Notch activation complies with the mosaic analysis performed in Drosophila, which revealed that shibire function is required in Notch-expressing cells receiving a lateral inhibition signal. It is proposed that monoubiquitination on a juxtamembrane lysine (K1749) and endocytosis occur after ligand-induced cleavage of the Notch extracellular domain by TACE. The data are in apparent contradiction with a previous model, according to which gamma-secretase cleavage occurs at the plasma membrane. However, the previous data can be reinterpreted in light of the new model. The previous study argued that the TACE-processing product of Notch (similar to the DeltaE construct used in this study) remains associated with the apical membrane in Nicastrin or PS mutant cells, and in WT cells only a small amount of this molecule can be found in endocytic vesicles. This result can be explained by the fact that ubiquitination is one of the limiting steps in Notch signaling, or that active PS is needed to direct the final steps of endocytosis of the ubiquitinated forms. Probably for the same reason, DeltaE is very poorly cleaved by gamma-secretase when overexpressed, and the ubiquitination event can hardly be detected. The current results are also in apparent contradiction with those of another study, which postulates that in Drosophila, PS-mediated proteolysis does not appear to require a particular sequence nor the presence of active dynamin. However, the assay used appears unusually sensitive, as it even detects the cleavage of a Notch molecule carrying a G1743V mutation of the gamma-secretase cleavage site, a mutation that prevents activation in most other assays and, when introduced into mice, gives rise to an almost perfect Notch1 null phenotype. Therefore, a leakage due to overexpression might in some cases be responsible for the activity detected. Experiments are in progress to test the effect of mutating the juxtamembrane lysine of Drosophila Notch (Gupta-Rossi, 2004).
Various papers have described monoubiquitination as a signal for internalization of receptors such as EGFR or glycine receptor. The results do not allow one to discriminate between ubiquitination triggering endocytosis or being concomitant with the first steps of endocytosis. However, the observations show a more internal localization of LLFF (the site of gamma-secretase cleavage) compared with the K1749R mutant, and endocytosis of the K1749R DeltaE mutant seems to be blocked at an earlier stage when compared with the WT or LLFF mutant. These data suggest that ubiquitination is necessary for late events driving Notch to compartments where gamma-secretase cleavage can occur (Gupta-Rossi, 2004).
The cleavage of Notch by presenilin (PS)/gamma-secretase is a salient example of regulated intramembrane proteolysis, an unusual mechanism of signal transduction. This cleavage is preceded by the binding of protein ligands to the Notch ectodomain, activating its shedding. It was hypothesized that the Notch ligands, Delta and Jagged, themselves undergo PS-mediated regulated intramembrane proteolysis. The ectodomain of mammalian Jagged is shown to be cleaved by an A disintegrin and metalloprotease (ADAM) 17-like activity in cultured cells and in vivo, similar to the known cleavage of Drosophila Delta by Kuzbanian. The ectodomain shedding of ligand can be stimulated by Notch and yields membrane-tethered C-terminal fragments (CTFs) of Jagged and Delta that accumulate in cells expressing a dominant-negative form of PS or treated with gamma-secretase inhibitors. PS forms stable complexes with Delta and Jagged and with their respective CTFs. PS/gamma-secretase then mediates the cleavage of the latter to release the Delta and Jagged intracellular domains, a portion of which can enter the nucleus. The ligand CTFs compete with an activated form of Notch for cleavage by gamma-secretase and can thus inhibit Notch signaling in vitro. The soluble Jagged intracellular domain can activate gene expression via the transcription factor AP1, and this effect is counteracted by the co-expression of the gamma-secretase-cleaved product of Notch, Notch intracellular domain. It is concluded that Delta and Jagged undergo ADAM-mediated ectodomain processing followed by PS-mediated intramembrane proteolysis to release signaling fragments. Thus, Notch and its cognate ligands are processed by the same molecular machinery and may antagonistically regulate each other's signaling (LaVoie, 2003).
The evolutionary conserved Notch signaling pathway is involved in cell fate specification and mediated by molecular interactions between the Notch receptors and the Notch ligands -- Delta, Serrate, and Jagged. Like Notch, Delta1 and Jagged2 are subject to presenilin (PS)-dependent, intramembranous 'gamma-secretase' processing, resulting in the production of soluble intracellular derivatives. Moreover, and paralleling the observation that expression of familial Alzheimer's disease-linked mutant PS1 compromises production of Notch S3/NICD, the PS-dependent production of Delta1 cytoplasmic derivatives are also reduced in cells expressing mutant PS1. These studies led to the conclusion that a similar molecular apparatus is responsible for intramembranous processing of Notch and it's ligands. To assess the potential role of the cytoplasmic derivative on nuclear transcriptional events, a Delta1-Gal4VP16 chimera was expressed and marked transcriptional stimulation of a luciferase-based reporter was demonstrated. These findings suggest that Delta1 and Jagged2 play dual roles as activators of Notch receptor signaling and as receptors that mediate nuclear signaling events via gamma-secretase-generated cytoplasmic domains (Ikeuchi, 2003).
Presenilins have been implicated in the genesis of Alzheimer's disease and in facilitating LIN-12/Notch activity during development. All presenilins have multiple hydrophobic regions that could theoretically span a membrane, and a description of the membrane topology is a crucial step toward deducing the mechanism of presenilin function. An eight-transmembrane-domain model has been proposed for presenilin, based on studies of the Caenorhabditis elegans SEL-12 presenilin. Experiments are described that support the view that two of the hydrophobic regions of SEL-12 function as seventh and eighth transmembrane domains. Human presenilin 1 behaves like SEL-12 presenilin when analyzed by these methods. These results provide additional experimental support for the eight-transmembrane-domain model of presenilin topology (Li, 1998).
The carboxyl terminus of presenilin 1 and 2 (PS1 and PS2) binds to the neuron-specific cell adhesion molecule telencephalin (TLN) in the brain. TLN (or ICAM-5) is a neuron- and region-specific member of the ICAM subfamily of intercellular adhesion molecules. TLN promotes dendritic outgrowth and contributes to long-term potentiation. PS1 deficiency results in the abnormal accumulation of TLN in a yet unidentified intracellular compartment. The first transmembrane domain and carboxyl terminus of PS1 form a binding pocket with the transmembrane domain of TLN. Remarkably, APP binds to the same regions via part of its transmembrane domain encompassing the critical residues mutated in familial Alzheimer's disease. These data surprisingly indicate a spatial dissociation between the binding site and the proposed catalytic site near the critical aspartates in PSs. They provide important experimental evidence to support a ring structure model for PS (Annaert, 2001).
Only a small fraction of TLN binds PS1 under steady-state conditions, indicating that the interaction is functional and not structural. PS1 is mainly restricted to pre-Golgi compartments in cultured neuronal cells, while TLN resides almost exclusively at the somatodendritic plasma membrane. Occasionally, some PS1-positive membranes closely juxtaposed to TLN positive patches were observed, probably at the level of focal contacts. However, PS1 deficiency causes striking alterations in the subcellular distribution of endogenous TLN, which becomes missorted and accumulates in large intracellular structures. TLN, a strongly developmentally regulated protein, is detected significantly earlier in PS1-/- neurons than in their wild-type counterparts. Also, the relative numbers of neurons expressing TLN at the cell surface increases more rapidly in the absence of PS1. Furthermore, because TLN accumulations are only seen in differentiated PS1-/- neurons and not at early stages, they apparently reflect a time-related cumulative effect of PS1 deficiency. This can easily be interpreted in a context of a defective functional PS1-TLN interaction, even if only small quantities of TLN interact with PS1 at any given point in time (Annaert, 2001).
Interestingly, TLN accumulates in intracellular structures and seems to cause a local reorganization of the subcortical actin cytoskeleton. A link is known to exist between the actin cytoskeleton and TLN. This interaction is important for intercellular adhesion believed to control neurite outgrowth in the telencephalon. Since Notch signaling is also implicated in neurite outgrowth and branching, it follows that PS1 apparently controls at least two pathways involved in neuritic arborization. It is therefore surprising that the absence of PS1 has so little effect on the overall morphology of mature neurons in culture. It is concluded that important compensating mechanisms must be at work and more subtle, yet-to-be discovered physiological alterations in neuritic outgrowth and/or synaptogenesis caused by the absence of PS1 are anticipated. In line with this hypothesis are recent observations in PS1 conditionally targeted mice. These mice do not show abnormalities in the Notch signaling pathway, but nevertheless display subtle cognitive deficits. Whether accumulation of TLN can explain this phenotype is an interesting possibility. It is to be noted that mice lacking TLN also display no abnormalities apart from changes in hippocampal LTP (Annaert, 2001).
The interaction of TLN with the C terminus of PS1 requires at least five amino acids (Val-829 toTrp-833) in the N-terminal part of the transmembrane region. This suggests that the hydrophobic PS1 C terminus can intrude into the lipid bilayer to interact with this part of the TLN transmembrane domain. Also for APP, the PS1 binding site is in the transmembrane domain, but located in the 11 amino acids (Thr-639 to Lys-649) situated at its C-terminal end. This extends previous work demonstrating that the cytoplasmic domain of APP and large parts of the ectodomain are not needed for PS1 binding. It is speculated that the binding region is crucial for the presentation of APP to the catalytic domain of gamma-secretase. In support of this conclusion, phenylalanine-scanning mutagenesis of this region significantly influenced gamma-secretase processing of APP. Also peptidomimetics that inhibit the gamma42 cleavage of APP include amino acids of this region. Most importantly, all known FAD-causing missense mutations in APP that shift the gamma-secretase cleavage toward Aß42 production are located within this short sequence. It seems likely that the FAD mutations may affect the binding of APP with PS1 and that, by doing so, they modulate the presentation of APP to gamma-secretase. Therefore, small compounds mimicking the binding sites in APP or in TLN, or binding selectively to the APP and not the TLN sequence, could possibly prevent the processing of APP by gamma-secretase (Annaert, 2001).
TLN was identified via two-hybrid screening using the C-terminal eight amino acids of PS1 as a bait. Residues Leu-460 to Ile-467 in PS1 thus define the minimal binding region with TLN. The interaction is more efficient when the whole C terminus of PS1 (or PS2) is used (Lys-429-Ile-467, and the corresponding sequence in PS2), which is not unexpected since the structure of a peptide in a protein is strongly influenced by neighboring sequences. Importantly, when the PS1 protein was scanned for additional binding sites, it was established that the first transmembrane region of PS1 (Val-82-Ser-102) determines a second important binding site for TLN and APP. Two domains at opposing sites in the PS1 sequence are therefore apparently involved in TLN and APP binding. These results suggest that certain type I membrane proteins bind via their transmembrane domain to a common binding pocket constituting the C-terminal domain and the first integral membrane domain of PS1 (Annaert, 2001).
The fact that the binding domains in PS1 are exceptionally well conserved among different species further corroborates the hypothesis that they are of major functional importance. Consistently, only few disease-linked mutations are found in these regions while some loss-of-function mutations in these domains are found in the PS homologs of C. elegans and Drosophila. If both domains comprise together a functional binding pocket, they should be closely juxtaposed in the lipid bilayer, suggesting a circular or ring-like structure for PS1. Although provocative, such a model supports recent findings that intramolecular associations between different domains of PS1, as well as cooperative interactions between both fragments, are important for the functionality of the PS complex. While other, more complicated models could be envisaged, the fact that the PS1Deltaexon 9, as well as other mutations that prevent endoproteolysis of PS1, maintains gamma-secretase cleavage of APP supports the model that both fragments remain closely associated in a ring-like structure. The model implies the possibility that the hydrophobic C terminus of PS1 can penetrate to different extents into the membrane, and a regulatory function is postulated for this part of PS1. Furthermore, the model suggests that the supposedly catalytic site in PS1 and the APP binding site are remote. This implies that binding and cleavage of substrate are two separate events. If PS1 is indeed the gamma-secretase, it has to be assumed that substrate presentation to the catalytic domain near the aspartate residues involves a refolding of the PS1 binding module into the interior of the ring structure. It is also possible that PSs, after binding substrates like APP(-CTF), Notch, or TLN, tag or transport these proteins for destination to a downstream compartment. The missorting of TLN in PS1-deficient neurons is at least compatible with this possibility as well (Annaert, 2001).
To date, the type I membrane proteins shown to bind PS can be subdivided in three functional classes. APP and Notch are gamma-secretase substrates, and their regulated intramembrane proteolysis depends critically on PSs. Nicastrin binds strongly to PSs, but appears to be a modulator of and not a substrate for gamma-secretase. Finally, cadherin and TLN are both cell adhesion proteins, and PS1 may modulate their correct cell membrane insertion and therefore their adhesive functions at the cell membrane. PS1 also regulates (to a certain extent) Wnt signaling via its interaction with ß-catenin (Annaert, 2001).
Therefore, the PSs are at the crossroads of several important signaling pathways, and the question is now whether PSs are a means of cross-talk between these pathways. By defining precisely the molecular domains involved in the interaction of PS1 with APP and TLN, a structural basis for further investigations of this question is provided. It is also clear that these binding sites provide novel potentially important targets for drug development in the fight against Alzheimer's disease (Annaert, 2001).
Presenilin: Evolutionary homologs part 2/3 | part 3/3 |
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