In animal development, numerous cell-cell interactions are mediated by the GLP-1/LIN-12/NOTCH family of transmembrane receptors. These proteins function in a signaling pathway that appears to be conserved from nematodes to humans. The aph-2 gene is a new component of the GLP-1 signaling pathway in the early Caenorhabditis elegans embryo, and proteins with sequence similarity to the APH-2 protein are found in Drosophila and vertebrates. During the GLP-1-mediated cell interactions in the C. elegans embryo, APH-2 is associated with the cell surfaces of both the signaling, and the responding, blastomeres. Analysis of chimeric embryos that are composed of aph-2+ and aph-2- blastomeres suggests that aph-2+ function may be provided by either the signaling or responding blastomere (Guotte, 2000).
aph-2 encodes a novel extracellular protein required for GLP-1-mediated signaling. Aph-2, termed Nicastrin in this study, 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 contain 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) is 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).
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).
Early embryonic cells in C. elegans embryos interact through a signaling pathway closely related to the Notch signaling pathway in Drosophila and vertebrates. Components of this pathway include a ligand, receptor, the presenilin proteins, and a novel protein, APH-2, that is related to the Nicastrin protein in humans. The aph-1 gene has been identified as a new component of the Notch pathway in C. elegans. aph-1, coding for a protein with cognates in Drosophila and mammals, encodes a novel, highly conserved multipass membrane protein. aph-1 and the presenilin genes share a similar function in that they are both required for proper cell-surface localization of APH-2/Nicastrin (Goutte, 2002).
APH-1 and related proteins are largely hydrophobic, with a common pattern of seven hydrophobic regions that are predicted to be membrane-spanning regions. None of the proteins contain predicted glycosylation sites, consistent with the idea that the majority of the protein is embedded within a cellular membrane. There are conserved, hydrophilic residues within the hydrophobic domains (Cys-9, Ser-45, Ser-50, Ser-130, His-183, Ser-213, His-214, Ser-257), suggesting that these residues may be critical for protein structure or interactions with other proteins. The aph-1(or28) mutation is predicted to replace a glycine residue with an aspartic acid residue within the fourth hydrophobic domain, and may therefore disrupt the topography of APH-1 in the membrane. The C. elegans APH-1 protein is unique in containing a hydrophilic 40-aa 'tail' at the C terminus. The aph-1(zu147) mutation results in a premature stop codon near the beginning of this hydrophilic tail. Because aph-1(zu147) appears to be a hypomorphic allele that retains some aph-1(+) activity, the nonconserved C-terminal tail may not be essential for APH-1 function. The only other multipass membrane proteins known to act in C. elegans Notch pathways are the presenilin proteins (Goutte, 2002).
Because of its characteristics as a membrane protein, it was asked whether the APH-1 protein might influence the expression or localization of cell-surface components of the Notch pathway in the early embryo. In wild-type 4-cell stage embryos, the ligand APX-1 is localized to the surface membrane of the posterior-most cell, where it contacts one of the two cells that express the receptor GLP-1. The APH-2 protein is associated with the surface membranes of all four cells. APX-1 and GLP-1 have the wild-type localization pattern in aph-1 mutant embryos; however, the APH-2 protein is mislocalized in all aph-1 mutant embryos examined. In aph-1 mutant embryos, APH-2 is not detectable on the cell surface, and is instead prominent in the cytoplasm of all four cells, where it is concentrated around the nucleus in a pattern characteristic of the endoplasmic reticulum. These results suggest that APH-1 facilitates the translocation of APH-2 to the cell surface (Goutte, 2002).
Presenilin proteins have been localized to the endoplasmic reticulum in some systems, and are essential for Notch signaling. Because the human orthologue of APH-2, Nicastrin, has been shown to associate with presenilins, it was asked whether presenilin function is required for APH-2 localization. Remarkably, presenilin-deficient embryos show an abnormal accumulation of APH-2 around the nucleus in an apparently identical pattern as that of aph-1 mutant embryos; these embryos had the wild-type pattern of GLP-1 localization. These results suggest that the presenilins and APH-1 may function together in an event that is a prerequisite for APH-2 to localize to the cell surface (Goutte, 2002).
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).
The presenilins and nicastrin, a type 1 transmembrane glycoprotein, form high molecular weight complexes that are involved in cleaving the beta-amyloid precursor protein (betaAPP) and Notch in their transmembrane domains. The former process (termed gamma-secretase cleavage) generates amyloid beta-peptide (Abeta), which is involved in the pathogenesis of Alzheimer's disease. The latter process (termed S3-site cleavage) generates Notch intracellular domain (NICD), which is involved in intercellular signalling. Nicastrin binds both full-length betaAPP and the substrates of gamma-secretase (C99- and C83-betaAPP fragments), and modulates the activity of gamma-secretase. Nicastrin is shown in this study to bind to membrane-tethered forms of Notch (substrates for S3-site cleavage of Notch), and, although mutations in the conserved 312-369 domain of nicastrin strongly modulate gamma-secretase, they only weakly modulate the S3-site cleavage of Notch. Thus, nicastrin has a similar role in processing Notch and betaAPP, but the 312-369 domain may have differential effects on these activities. In addition, the Notch and betaAPP pathways do not significantly compete with each other (Chen, 2001).
Nicastrin acts as a key regulator for presenilin (PS)-mediated gamma-secretase cleavage of beta-amyloid precursor protein by forming a functional complex with PS1 and PS2. Both TNF-alpha and IL-1, aberrantly produced by activated microglia and astrocytes, play a role in amyloidogenesis and neurodegeneration in the brains of Alzheimer's disease (AD) patients, while BDNF synthesized chiefly by neurons has been found to be substantially reduced in AD brains. To investigate the constitutive and cytokine/neurotrophic factor-regulated expression of nicastrin in human neural cells, its mRNA levels were studied by RT-PCR and Northern blot analysis in SK-N-SH neuroblastoma cells, IMR-32 neuroblastoma cells, U-373MG astrocytoma cells, and NTera2 teratocarcinoma-derived differentiated neurons (NTera2-N) following exposure to TNF-alpha, IL-1beta, BDNF, dibutyryl cyclic AMP, or phorbol 12-myristate 13-acetate. Nicastrin mRNA expression was identified in all human neural and nonneural cell lines and tissues examined. However, the levels of nicastrin mRNA were unaltered in SK-N-SH, IMR-32, U-373MG, and NTera2-N cells by exposure to the factors tested, and unchanged in NTera2 cells during retinoic acid-induced neuronal differentiation. These results indicate that nicastrin mRNA is expressed constitutively in human neural cell lines, where its expression is not regulated at the transcriptional level by a battery of cytokines and growth/differentiation factors that are supposed to be involved in amyloidogenesis, neurodegeneration or neuroprotection in AD brains (Satoh, 2001).
Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. A genetic screen was set up based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. Clones were selected deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. Two of these clones were examined and previously undescribed mutations were identified in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired (Olry, 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).
The gamma-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this 'proteasome of the membrane' are unknown. A new interaction partner of nicastrin, the retrieval receptor Rer1p, has been characterized. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of gamma-secretase subcomplexes and, concomitantly, total cellular gamma-secretase activity. Rer1p is a novel limiting factor that negatively regulates gamma-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. It is concluded that total cellular gamma-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value (Spasic, 2007).
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