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2 National Cancer Institute, Frederick, MD 21702
Address correspondence to M.E. Fortini, National Cancer Institute, Bldg. 560, Rm. 22-12, Frederick, MD 21702. Tel.: (301) 846-7599. Fax: (301) 846-1666. E-mail: fortini{at}ncifcrf.gov
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Abstract |
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Key Words: presenilin; Pen-2; Notch; Drosophila; amyloid
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Introduction |
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Psn itself is synthesized as an immature holoprotein that undergoes endoproteolysis to generate NH2- and COOH-terminal fragments, which are thought to associate to form the active enzyme (Thinakaran et al., 1996; Ratovitski et al., 1997; Levitan et al., 2001). The trafficking and processing events associated with maturation of Psn and its substrates have complicated efforts to understand the assembly of -secretase and its proteolytic activation. Significantly, only a fraction of Psn is endoproteolyzed and assembled into these high mol wt complexes, with the majority of the Psn holoprotein instead being rapidly degraded (Ratovitski et al., 1997; Thinakaran et al., 1997).
Recent work has identified three additional putative -secretase components, termed nicastrin (Nct), Aph-1, and Pen-2. Nct is a type I integral membrane protein that associates with Psn and COOH-terminal fragments of APP and Notch (Yu et al., 2000; Chen et al., 2001). Aph-1 and Pen-2 are polytopic membrane proteins containing seven and two predicted transmembrane segments, respectively, and were identified through genetic screens in Caenorhabditis elegans (Francis et al., 2002; Goutte et al., 2002). Many questions remain about the roles of these components in
-secretase assembly, trafficking, and enzymatic activation. The fact that only a fraction of Psn is endoproteolyzed has led to the proposal that limiting cellular factors regulate the amount of active
-secretase that is produced (Ratovitski et al., 1997; Thinakaran et al., 1997). Are Nct, Aph-1, and Pen-2 these postulated limiting factors, and are they, together with Psn, sufficient for functional
-secretase assembly? Which of these components are associated with mature
-secretase, and do any display specific roles in
-secretase complex assembly or function? Here, we investigate the activities of the four putative components of Drosophila
-secretase in the context of Notch signaling and proteolysis. We identify aph-1 mutant flies, which exhibit defects in Notch function that are indistinguishable from Drosophila Psn and nct mutants. In fly Schneider-2 (S2) cells, high level accumulation of mature Psn and Pen-2, as well as increased
-secretase cleavage of Notch, are only observed if all four putative
-secretase components are coexpressed in S2 cells. In contrast to other components, Aph-1 is stabilized by Nct expression, but destabilized by the additional coexpression of Psn. We propose an assembly pathway in which Aph-1 and Nct associate in a subcomplex that acts early in
-secretase complex formation to stabilize Psn holoprotein, and in which Pen-2 is required for endoproteolysis of Psn holoprotein.
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Results and discussion |
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The postembryonic lethality of aph-1 mutants indicated that an embryonic requirement for Aph-1 in Notch signaling might be masked by maternally deposited protein. Therefore, we generated adult females bearing aph-1 mutant germline clones, and found that embryos lacking both maternal and zygotic Aph-1 activity exhibit a Notch-like hyperplasia of the embryonic nervous system (Fig. 1, A and B). Similarly, aph-1 mutant wing discs contain clusters of supernumerary sensory organ precursor (SOP) cells in addition to an overall developmental arrest phenotype, as seen in other neurogenic mutants (Fig. 1, I and J). To examine adult tissue phenotypes, we induced homozygous mutant aph-1 somatic tissue clones in heterozygous hosts. Numerous phenotypes characteristic of impaired Notch function were observed, including wing notching and bristle loss (Fig. 1, CH). Together, these results demonstrate that Aph-1 is required for Notch signaling throughout development, and that aph-1 mutants show phenotypes indistinguishable from Psn and nct mutants.
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Aph-1 and Pen-2 are required for Psn maturation in Drosophila cells
Using a form of Psn with a myc epitope inserted in the hydrophilic loop to detect the COOH-terminal subunit, or a form with an NH2-terminal myc tag, we observed loss of both the mature NH2- and COOH-terminal fragments, but no effect on the immature Psn holoprotein when cells were treated with double-stranded RNA (dsRNA) directed against either aph-1 or pen-2 (Fig. 2). These results are similar to those reported for aph-1 and pen-2 in C. elegans, Drosophila, and mammalian cells (Francis et al., 2002; Lee et al., 2002; Steiner et al., 2002; Gu et al., 2003; Luo et al., 2003). Our result with pen-2 RNA-mediated interference (RNAi) was also similar to that observed by Takasugi et al. (2003) in Drosophila cells, but their work found that aph-1 RNAi leads to loss of both Psn holoprotein and cleaved fragments. This discrepancy is likely to reflect the fact that their approach used a relatively weak promoter to drive Psn expression, such that all newly synthesized Psn holoprotein was able to enter the Aph-1dependent early steps of -secretase assembly. Under these conditions, depletion of Aph-1 would eliminate all the stabilized Psn holoprotein and its derivative fragments. Our use of a stronger promoter presumably results in an excess of Psn holoprotein that is unable to enter this stabilization pathway, and which, therefore, is insensitive to removal of Aph-1 or Nct. Interestingly, although Takasugi et al. (2003) found that nct RNAi also abolished all Psn holoprotein in their cell culture assay, a previous in vivo genetic analysis detected Psn immunoreactivity in early compartments of the secretory pathway of nct mutant flies (Chung and Struhl, 2001), suggesting that physiological levels of Psn expression might be high enough to allow some Psn holoprotein to accumulate in the absence of these
-secretase cofactors.
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S2 cells were transfected with constructs encoding stepwise combination of the four -secretase components. No massive overproduction of Psn holoprotein or COOH-terminal fragments was observed in samples involving up to three coexpressed proteins. However, coexpression of all four proteins together results in high levels of mature
-secretase, as indicated by dramatically elevated levels of cleaved Psn COOH-terminal fragments and tagged Pen-2 (Fig. 3, A and B). These effects were paralleled by a significant increase in the specific Notch p120 cleavage attributed to
-secretase, implying an elevated production of functional
-secretase (Fig. 4). Nct levels remain relatively constant in all samples, including those involving coexpression of all four proteins (Fig. 3 B). However, Aph-1 accumulates to high levels only when coexpressed with Nct or Nct with Pen-2, as described later (Fig. 3 B).
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Given the technical difficulties associated with producing purified multipass membrane proteins such as Psn, Pen-2, and Aph-1 without relying on cells or microsomes, our coexpression strategy approximates a cellular "reconstitution" of purified -secretase components, with the caveat that this approach cannot rigorously exclude the potential involvement of unknown cellular factors. Using a similar stepwise coexpression approach, Takasugi et al. (2003) have recently reported similar results that coexpression of Drosophila Aph-1, Pen-2, and Nct increases levels of Psn fragments and increases APP
-secretase cleavage. Similarly, Edbauer et al. (2003) have recently reported equivalent results by coexpressing mammalian
-secretase components in yeast.
Effects of Aph-1 and Pen-2 indicate different roles in -secretase complex assembly
Aph-1 does not accumulate proportionally when all four putative -secretase components are coexpressed, suggesting that Aph-1 might be absent from the mature complex. Interestingly, coexpression of Aph-1 with Psn and Nct (without Pen-2) results in a moderate increase in both the Psn holoprotein as well as its processed fragments (Fig. 3, A and B). Conversely, coexpression of Pen-2 with Psn and Nct (without Aph-1) causes no discernible effect relative to coexpression of just Psn and Nct (Fig. 3, A and B). Because addition of Pen-2 to the Aph-1/Psn/Nct combination results in strong accumulation of the Psn COOH-terminal fragment with no apparent increase in Psn holoprotein, the simplest interpretation of these effects is that Aph-1 is important for determining the amount of Psn holoprotein that is stabilized and enters the maturation pathway, whereas Pen-2 is involved in subsequent Psn endoproteolysis. Our results are consistent with recent works showing that Aph-1 acts early during
-secretase assembly in the initial stabilization of Psn holoprotein, and that Pen-2 is required for holoprotein endoproteolysis (Gu et al., 2003; Luo et al., 2003; Takasugi et al., 2003).
Aph-1 and Nct might associate in a subcomplex
Another reliably observed effect involves the dramatic over-accumulation of Aph-1 when coexpressed together with Nct in the absence of Psn (Fig. 3, B and C). All other pairwise coexpression combinations involving Aph-1 fail to show significantly elevated levels of Aph-1. We observed strong up-regulation of Aph-1 by Nct using two different tagged versions of Aph-1 (Fig. 3 B and Fig. 5 A), and we also determined that it does not occur with Nct304333 and thus requires functional Nct (Fig. 3 C). Although Nct strongly enhances Aph-1 accumulation, this effect disappears if Psn is also present, but it is unaffected by Pen-2. It should be noted that these effects were not observed in a related paper by Takasugi et al. (2003). This discrepancy might be due to the fact that their work relied on coexpression of tagged Nct-V5 and tagged Aph-1-flag, similar to coexpression conditions under which we failed to detect strong Aph-1Nct stabilizing interactions due to epitope tag interference problems (Fig. 5 A).
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Concluding remarks
Our finding that coexpression of Psn, Nct, Aph-1, and Pen-2 leads to high levels of mature, functional -secretase, together with similar recent papers (Edbauer et al., 2003; Takasugi et al., 2003), suggests that these four proteins are likely to be the major limiting factors for
-secretase assembly, and that true biochemical reconstitution of
-secretase using purified proteins and membranes is feasible. A new result from our analysis is that Aph-1 and Nct appear to associate in a subcomplex that functions early in
-secretase assembly. We have incorporated our results with those of recent papers into a hypothetical model for
-secretase assembly and activation (Fig. 5 B). Further analysis will be required to test this model and to define more precisely the biochemical activities of each
-secretase component.
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Materials and methods |
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Immunohistology
Immunohistology of Drosophila tissues was performed as described previously (Ye and Fortini, 1998; Hu et al., 2002) using antibodies as follows: 1:200 rat anti-ELAV mAb 7E8A10, 1:30 mouse anti-Scabrous mAb sca1 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA).
S2 cell experiments
The aph-1 and pen-2 genes were obtained by PCR from cDNA libraries and used for dsRNA preparation and protein expression. Epitope-tagged forms were constructed in which V5 and flag tags were inserted at the COOH termini of Aph-1 and Pen-2. The Psn cDNA (Ye and Fortini, 1998) was modified to insert myc tags after Psn residues I26 or S412. The nct cDNA (Hu et al., 2002) was used to express untagged Nct, and a form was made with two myc tags at the COOH terminus. All cDNAs were subcloned into the pIZ-V5/His vector (Invitrogen). S2 transfections and RNAi were performed as described previously (Hu et al., 2002), using dsRNA for nct (+6741448), aph-1 (+71412) and pen-2 (+96400).
Western immunoblot analyses of S2 cells were performed as described previously (Ye et al., 1999; Hu et al., 2002). Gel loading was normalized according to total protein concentrations; some blots were also examined for levels of ß-tubulin or expression of cotransfected pIZ-GFP for loading and transfection controls. Western blots were probed with 1:1,000 mouse anti-Notch mAb C17.9C6, and anti-myc mAb (Sigma-Aldrich), anti-flag mAb (Upstate Biotechnology), anti-V5 mAb (Invitrogen), anti-GFP mAb (CLONTECH Laboratories, Inc.), or anti-ß-tubulin mAb (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) at recommended dilutions.
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Acknowledgments |
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This work was supported by intramural funding from the the National Cancer Institute and by grant RO1 AG14583 from the National Institute on Aging (to M.E. Fortini).
Submitted: 2 April 2003
Revised: 21 April 2003
Accepted: 21 April 2003
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References |
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