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Keywords:

  • Alzheimer's disease;
  • nicastrin;
  • APH-1;
  • presenilin;
  • PEN-2;
  • γ-secretase

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Antibodies
  5. cDNA constructs
  6. Cell culture, cell lines, and transfections
  7. Protein analysis
  8. Trypsin-resistance assay
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

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

Abbreviations used

amyloid-β peptide

AD

Alzheimer's disease

AICD

APP intracellular domain

APH-1

anterior pharynx-defective-1

APP

β-amyloid precursor protein

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

CTF

C-terminal fragment

HEK

human embryonic kidney

Nct

nicastrin

NTF

N-terminal fragment

PEN-2

presenilin enhancer-2

PS

presenilin

siRNA

small interfering RNA

RNAi

RNA interference

wt

wild-type

Alzheimer's disease (AD) is a frequent disorder in an aging society. An invariant pathological feature is the deposition of amyloid β-peptide (Aβ) in senile plaques. Aβ is generated by proteolytic processing of the β-amyloid precursor protein (APP) via two secretases, β-secretase and γ-secretase. β-Secretase is a well-characterized aspartyl protease with many expected biochemical features characteristic for this class of proteases (Vassar 2001). In contrast, γ-secretase is a rather unusual aspartyl protease. γ-Secretase activity is associated with a high molecular mass complex (Li et al. 2000; Haass and Steiner 2002; De Strooper 2003). Either of the two homologous presenilins, PS1 and PS2, apparently comprises the catalytic core of this complex. PSs contain two critical aspartyl residues in their putative catalytically active core (Steiner et al. 1999; Wolfe et al. 1999). One of them is embedded within a conserved GXGD (with X being a variable amino acid) motif also occurring in bacterial type 4 prepilin peptidases and the signal peptide peptidases and their homologues (Steiner et al. 2000; Weihofen et al. 2002). Biochemical purification of this complex led to the identification of the second γ-secretase complex component, termed nicastrin (Nct) (Yu et al. 2000). Genetic screens for enhancers of a PS-dependent Notch phenotype in Caenorhabditis elegans led to the identification of two additional components: APH-1 (anterior pharynx-defective-1) and PEN-2 (PS-enhancer-2) (Francis et al. 2002; Goutte et al. 2002). These four components are sufficient for reconstituting γ-secretase complex formation, PS endoproteolysis, as well as Aβ and AICD (APP intracellular domain) production in Saccharomyces cerevisiae, an organism that lacks any endogenous γ-secretase (Edbauer et al. 2003). Individual overexpression of Nct, APH-1 or PEN-2 can increase γ-secretase activity (Marlow et al. 2003; Murphy et al. 2003) suggesting excess amounts of remaining γ-secretase components. Even higher γ-secretase activity is obtained when all four components are co-expressed in cultured mammalian and fly cells (Hu and Fortini 2003; Kim et al. 2003; Kimberly et al. 2003; Marlow et al. 2003; Takasugi et al. 2003).

PS expression and γ-secretase activity are regulated in a coordinated manner. If one of the components is missing, expression of the remaining components is reduced or as in the case of Nct fails to maturate (Edbauer et al. 2002; Francis et al. 2002; Kimberly et al. 2002; Lee et al. 2002; Leem et al. 2002; Steiner et al. 2002; Chen et al. 2003; Herreman et al. 2003; Li et al. 2003a; 2003b; Luo et al. 2003; Takasugi et al. 2003). In addition, in the absence of PEN-2 (and probably Nct as well (Li et al. 2003a), PS accumulates as a stabilized holoprotein (Kim et al. 2003; Luo et al. 2003; Takasugi et al. 2003). Likewise, stabilized PS holoprotein accumulates upon overexpression of Nct and APH-1 (Kim et al. 2003; Luo et al. 2003; Takasugi et al. 2003). Upon additional overexpression of PEN-2, the stabilized PS holoprotein is efficiently endoproteolyzed into its fragments (Hu and Fortini 2003; Kim et al. 2003; Luo et al. 2003; Takasugi et al. 2003) and full γ-secretase activity is obtained. Thus, APH-1 together with Nct appears to be a stabilizer of the PS holoprotein, whereas PEN-2 is required to initiate its endoproteolysis.

The findings described above suggest distinct steps in the assembly of the γ-secretase complex and putative precomplexes composed of the PS holoprotein, Nct, and APH-1 (Takasugi et al. 2003) or Nct and APH-1 (LaVoie et al. 2003) and probably PEN-2 (Hu and Fortini 2003) have been suggested recently. In the latter case, PS seems to displace APH-1 from the mature complex (Hu and Fortini 2003). Nct may be the first and initial binding partner of γ-secretase complex components, as in the absence of PS1/2 or PEN-2 its expression is not severely reduced but it rather remains as an immature protein within the endoplasmic reticulum (ER) (Edbauer et al. 2002; Kimberly et al. 2002; Leem et al. 2002; Yang et al. 2002; Herreman et al. 2003). Nct is inserted into the endoplasmic reticulum and matures by complex glycosylation during its trafficking through the secretory pathway (Yu et al. 2000; Edbauer et al. 2002; Kaether et al. 2002; Kimberly et al. 2002; Leem et al. 2002; Yang et al. 2002). Elimination of PS, APH-1, or PEN-2 abolishes maturation of Nct by complex glycosylation (Edbauer et al. 2002; Kimberly et al. 2002; Lee et al. 2002; Leem et al. 2002; Steiner et al. 2002; Chen et al. 2003; Gu et al. 2003; Herreman et al. 2003). However, glycosylation is not required for Nct function within the γ-secretase complex (Herreman et al. 2003; Shirotani et al. 2003). During its maturation, Nct undergoes a major structural alteration within its large luminal domain (Shirotani et al. 2003). Deletion analysis revealed that not only the highly conserved DYIGS domain (Yu et al. 2000) but that rather the entire luminal domain is required for Nct activity within the complex (Shirotani et al. 2003). Thus, folding of the Nct ectodomain is required for γ-secretase complex formation and activity (Shirotani et al. 2003). In addition, the transmembrane domain of Nct appears to be necessary for its assembly into the maturating γ-secretase complex (Capell et al. 2003; Morais et al. 2003). However, these studies did not allow the identification of selective point mutants of Nct, which may affect γ-secretase function. We thus mutated evolutionary conserved single amino acids and analysed the resulting Nct variants for in vivo function. Surprisingly, we identified mutants, which selectively bound to APH-1 but lost their capability to interact with and to stabilize PS and PEN-2. Consequently, these mutants failed to rescue γ-secretase function. Consistent with this observation, we also observed a stable Nct/APH-1 interaction in cells derived from PS1/2 double knock-out mice. Taken together, our data demonstrate an interaction of immature Nct with APH-1, which may occur as an initial interaction during the assembly of the γ-secretase complex. The failure of the Nct mutants to bind and stabilize PS and PEN-2 suggests that binding of the latter two complex components critically depends on the integrity of the Nct ectodomain.

Antibodies

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Antibodies
  5. cDNA constructs
  6. Cell culture, cell lines, and transfections
  7. Protein analysis
  8. Trypsin-resistance assay
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

The polyclonal and monoclonal antibodies against the large cytoplasmic loop domain of PS1 (3027), the PS1 N-terminus (PS1N), the large cytoplasmic loop domain of PS2 (BI.HF5c), PEN-2 (1638), and the APP C-terminus (6687) were described previously (Edbauer et al. 2002; Steiner et al. 2002 and citations therein). The polyclonal antibody 9424 was generated against the APH-1aL C-terminus (residues 245–265) and affinity-purified. The anti-APH1aL antibody O2C2 was described previously (Gu et al. 2003). The polyclonal antibody N1660 against the C-terminus of Nct and SPA-860 against calnexin were obtained from Sigma (St. Louis, MO, USA) and StressGen (Victoria, BC, Canada), respectively.

cDNA constructs

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Antibodies
  5. cDNA constructs
  6. Cell culture, cell lines, and transfections
  7. Protein analysis
  8. Trypsin-resistance assay
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

Mutant Nct cDNAs were constructed by oligonucleotide-directed mutagenesis using PCR and cloned into pcDNA6. All constructs were verified by DNA sequencing. Human APH-1aL encoding the 265 amino acid long splice variant (Lee et al. 2002) was amplified from a human brain Matchmaker cDNA library (Clontech, Palo Alto, CA, USA) and subcloned into pcDNA3.1/Zeo(+) (Invitrogen Carlsbad, CA, USA).

Cell culture, cell lines, and transfections

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Antibodies
  5. cDNA constructs
  6. Cell culture, cell lines, and transfections
  7. Protein analysis
  8. Trypsin-resistance assay
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

HEK293 cells and mouse embryonic fibroblast cells were cultured as described (Edbauer et al. 2002). The stable Nct knock-down cell line overexpressing Swedish (sw) mutant APP (sw/NctRNAi) has been described previously (Shirotani et al. 2003). This cell line was stably transfected with the indicated wt and mutant Nct constructs or the parental vector (pcDNA6) using LipofectAmine 2000 (Invitrogen) according to the instructions of the manufacturer and single cell clones were isolated by selection for blasticidin (10 µg/mL) resistance. For the experiments shown in Fig. 2(a), individual single cell clones, which express different amounts of Nct were used.

image

Figure 2. Immature Nct and APH-1aL stabilize each other. (a) Dose-dependence of APH-1aL levels. Membrane proteins from independent single cell clones of the indicated HEK293 Nct knock-down cells stably transfected with wt Nct, or the Nct C248S, E333Q G339A mutant variants expressing increasing amounts of immature Nct were analysed for levels of Nct, APH-1aL, PS1 NTF, and PEN-2 by immunoblotting with antibodies N1660 (Nct), 9424 or O2C2 (APH-1aL), PS1 NTF (PS1N) and 1638 (PEN-2). (b) Dose-dependence of immature Nct levels. HEK293 Nct knock-down cells stably transfected with either wt Nct or the Nct C248S, G339A mutant variants were transiently transfected with increasing amounts of APH-1aL cDNA. Cell lysates were analysed for levels of Nct, APH-1aL, PS1 NTF and PEN-2 by immunoblotting with antibodies N1660 (Nct), 9424 (APH-1aL), PS1 NTF (PS1N) and 1638 (PEN-2). Note that endogenous APH-1aL is below the detection limit in cell lysates.

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Protein analysis

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Antibodies
  5. cDNA constructs
  6. Cell culture, cell lines, and transfections
  7. Protein analysis
  8. Trypsin-resistance assay
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

Membrane fractions of HEK293 cells were obtained by ultracentrifugation of postnuclear supernatant fractions from cell homogenates that were prepared as described (Sastre et al. 2001). For direct immunoblot analysis, membrane fractions were solubilized with STEN-lysis buffer [50 mm Tris (pH 7.6), 150 mm NaCl, 2 mm EDTA, 1% NP-40, protease inhibitors (Sigma)]. In addition, cell lysates that were prepared as described previously (Shirotani et al. 2003) were used for direct immunoblot analysis where indicated. For co-immunoprecipitation analysis, membrane fractions were solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-lysis buffer [2% CHAPS, 150 mm sodium citrate (pH 6.4), protease inhibitors (Sigma)], subjected to a clarifying spin by ultracentrifugation, diluted with three volumes of CHAPS-wash buffer [0.5% CHAPS, 150 mm sodium citrate (pH 6.4), protease inhibitor cocktail (Sigma)] and incubated with antibody N1660 and protein G-Sepharose for 2 h at 4°C. Following two washes in CHAPS-wash buffer, the immunoprecipitates were subjected to immunoblot analysis.

Trypsin-resistance assay

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Antibodies
  5. cDNA constructs
  6. Cell culture, cell lines, and transfections
  7. Protein analysis
  8. Trypsin-resistance assay
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

CHAPS-extracted membrane proteins were incubated with 100 µg/mL trypsin in 150 mm sodium citrate (pH 6.4), 5 mm EDTA, 5 µg/mL pepstatin for 30 min at 4°C and were subjected to immunoblot analysis (Shirotani et al. 2003).

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Antibodies
  5. cDNA constructs
  6. Cell culture, cell lines, and transfections
  7. Protein analysis
  8. Trypsin-resistance assay
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

In order to further define the sequence requirements for Nct function, we generated mutations of amino acid residues in the human Nct ectodomain, which are conserved in the Nct orthologs from mouse, zebrafish, Drosophila and C. elegans(Fig. 1). To avoid effects on folding of the luminal domain (Shirotani et al. 2003) isosteric point mutations were created where possible. We chose to mutate Cys248 because it is one of four equally spaced cysteine residues that are potentially important for Nct function (Yu et al. 2000). Pro262 was mutated because it may affect Nct structure and Asp283 as well as Glu333 were mutated because these residues are part of a domain that is conserved in the aminopeptidase/transferrin receptor superfamily (Fagan et al. 2001; Mushegian 2002). Although aminopeptidase activity has been ruled out for Nct (Fergani et al. 2001), the aminopeptidase/transferrin receptor-homologous domain in Nct may have retained an important function as a protein–protein interaction domain. Interestingly, Glu333 is located close to the highly conserved DYIGS motif, which was proposed to be important for γ-secretase function (Yu et al. 2000). To study the potential functional importance of this motif in more detail we mutated Gly339. In addition, we further analysed the previously described DY336/337 mutant (Yu et al. 2000). The corresponding cDNA constructs were stably transfected into human embryonic kidney 293 (HEK293 cells) stably expressing Swedish mutant APP and a pSUPER-based Nct-1045 siRNA (small interfering RNA)-encoding vector, which significantly knocks-down endogenous Nct expression by RNA interference (RNAi) (Shirotani et al. 2003). Consistent with our previous findings (Shirotani et al. 2003), knocking-down the expression levels of endogenous Nct led to dramatically reduced expression levels of PS1 and PS2 fragments, PEN-2, and of APH-1aL (Lee et al. 2002), which we investigated as an APH-1 archetype (Fig. 1b; lane 2). Coordinated down-regulation of these γ-secretase complex components led to the accumulation of APP C-terminal fragments (APP CTFs; Fig. 1b; lane 2) thus confirming the essential role of Nct for γ-secretase function (Edbauer et al. 2002; Shirotani et al. 2003). All aspects of this loss of function phenotype were rescued when a wt Nct cDNA construct containing RNAi-resistant mutations was expressed (Fig. 1b; lane 3). Similarly, when the point mutants P262L, D283N, or the DY/AA mutants were expressed, PS, PEN-2 and APH-1aL levels were largely restored (Fig. 1b). In addition, maturation of the mutant Nct variants was observed although as expected, excess amounts of Nct remained due to its overexpression immature (Fig. 1b) (Shirotani et al. 2003). Fully mature mutant Nct variants were resistant to trypsin treatment (Fig. 1c), suggesting that they were incorporated into an active γ-secretase complex (Shirotani et al. 2003). Moreover, upon expression of these Nct mutant variants the accumulation of APP CTFs was abolished (Fig. 1b) demonstrating restoration of normal γ-secretase function. In contrast maturation of the C248S, E333Q, and G339A Nct variants was severely impaired if not abolished (Fig. 1b) and the immature mutant Nct species were sensitive to trypsin (Fig. 1c). Consequently, they failed to restore γ-secretase function as monitored by the accumulation of APP CTFs (Fig. 1b). Likewise, they did not restore wt levels of PS1 and PS2 fragments and PEN-2. Surprisingly, all three Nct variants restored APH-1aL expression at least to some extent as compared to the Nct knock-down (Fig. 1b). This suggests that certain mutant Nct variants stabilize APH-1aL expression independent of PS and PEN-2 and indicate that immature Nct can stably associate with APH-1.

image

Figure 1. Identification of Nct mutants, which selectively interact with APH-1aL. (a) Schematic representation of wt Nct and the Nct point mutants generated. SP denotes the putative signal peptide and TM the transmembrane domain. Dotted boxes indicate conserved regions including the DYIGS motif-containing region (Yu et al. 2000). Potential glycosylation sites are indicated with black circles. The D336A/Y337A double mutation is abbreviated DY/AA. (b) PS- and PEN-2-independent stabilization of APH-1aL by the C248S, E333Q and G339A Nct mutants. HEK293 cells stably co-expressing Swedish mutant APP (sw) and Nct-1045 siRNA were stably transfected with the indicated cDNA constructs encoding wt Nct, Nct point mutants (both harbouring silent mutations to escape RNAi) or a vector control (pcDNA6). Membrane proteins were analysed for levels of Nct [mature (m) and immature (im) forms], PS1 and PS2 CTF, PEN-2, APH-1aL and calnexin as control by immunoblotting with antibodies N1660 (Nct), 3027 (PS1 CTF), BI.HF5c (PS2 CTF), 1638 (PEN-2), 9424 (APH-1aL) and SPA-860 (calnexin). APP CTFs, generated by γ- and α-secretase were analysed from cell lysates by immunoblotting with antibody 6687. Note that a very weak restoration of PS1 levels was observed with the E333Q mutant (see also Fig. 2a). The asterisk denotes the phosphorylated form of the PS1 CTF (Walter et al. 1997). (c) Trypsin-sensitivity of the C248S, E333Q and G339A mutant Nct variants. Membrane proteins from CHAPS-extracted HEK293 cells stably transfected with wt Nct and the indicated Nct mutants were treated with (+) or without (–) 100 µg/mL trypsin and analysed for Nct by immunoblotting with antibody N1660.

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To further confirm this finding, we investigated whether the stabilization of APH-1aL expression is dependent on the expression levels of the mutant Nct variants. Independent clonal lines of wt Nct, the C248S, E333Q or the G339A mutants expressing increasing amounts of immature Nct were investigated for a dose–response of APH-1aL levels. As shown in Fig. 2a, the Nct mutants displayed a dose-dependent increase of APH-1aL but not of the PS1 NTF and PEN-2, further suggesting that the mutants can only stably interact with APH-1aL and fail to interact with PS and PEN-2. Thus, the levels of available amounts of APH-1 can apparently be increased by stabilization with excess amounts of immature mutant Nct. In contrast, no dose–response of APH-1aL levels was observed with the wt Nct expressing clones (which express similar amounts of mature Nct) suggesting that all available endogenous APH-1 is already stabilized by complex formation with the other complex partners (Fig. 2a).

On the other hand, when increasing amounts of APH-1aL cDNA were transiently transfected in the wt Nct, C248S or G339A mutant cell lines expressing large excess amounts of free immature Nct, the immature Nct was stabilized in a dose-dependent manner by APH-1aL in all cases including wt Nct (Fig. 2b). Again, no dose-dependent stabilization of the PS1 NTF and PEN-2 levels was observed. Thus, excess levels of free immature Nct can be increased by stabilization only with increased levels of APH-1 (Fig. 2b). Taken together, immature Nct and APH-1 can stabilize each other.

We thus investigated binding of these two γ-secretase complex components in cells with severely reduced PS1 and PS2 fragments and PEN-2 levels by co-immunoprecipitation experiments. CHAPS-extracted membrane proteins, isolated from the cell lines described above, expressing either wt Nct or the Nct mutants C248S, E333Q, G339A were immunoprecipitated with a Nct antibody and analysed for binding of APH-1aL. Wt Nct co-immunoprecipitated with PS1 and PS2 fragments, PEN-2 and APH-1aL (Fig. 3a; lane 2). Interestingly, the immature Nct variants C248S, E333Q and G339A bound to APH-1aL (Fig. 3a). In contrast, if present, only very minor amounts of PS1 and PS2 fragments and PEN-2 were coimmunopecipitated (Fig. 3a; compare lanes 3–5 with lane 1). Thus, immature Nct is apparently able to bind to APH-1aL independently of PS1 and PS2 fragments or PEN-2. If that was the case, one should expect that APH-1aL levels are not as strongly reduced by a PS1/2 double knock-out, unlike in the case of PEN-2. To prove this, membrane proteins from mouse embryonic fibroblast cells derived from a PS1/PS2 gene knock-out or wt controls (Herreman et al. 2000) were immunoblotted with antibodies to the γ-secretase complex components. As expected, Nct failed to maturate in the double knock-out cells (Kimberly et al. 2002; Leem et al. 2002; Steiner et al. 2002; Chen et al. 2003) and PEN-2 levels were significantly reduced (Fig. 3b) (Steiner et al. 2002; Luo et al. 2003; Nyabi et al. 2003). However, APH-1aL levels remained largely unaffected (Fig. 3b). In contrast, when Nct was knocked-down via an RNAi approach, PEN-2 and APH-1aL were both severely reduced (Fig. 1b; lane 2) suggesting that APH-1aL requires the presence of Nct for its expression. Co-immunoprecipitation analysis of CHAPS-extracted membrane proteins from the PS1/PS2 double knock-out cells with an anti-Nct antibody revealed binding of immature Nct and APH-1aL in the absence of PS1/PS2 and PEN-2 (Fig. 3c). This result suggests that endogenous, immature wt Nct stably associates with APH-1aL independent of PS1/2 or PEN-2.

image

Figure 3. Complex formation of immature Nct with APH-1aL. (a) PS- and PEN-2-independent complex formation of immature Nct C248S, E333Q and G339A mutants with APH-1aL. CHAPS-extracted membrane proteins from HEK293 cells transfected with the indicated constructs were immunoprecipitated with antibody N1660 (Nct) and analysed by immunoblotting as in Fig. 1. The asterisk denotes the phosphorylated form of the PS1 CTF (Walter et al. 1997). (b) APH-1aL is stabilized in the absence of PS and PEN-2. Membrane proteins from PS1/2+/+ and PS1/2–/– mouse embryonic fibroblast cells were analysed for levels of Nct, PS1 CTF, PEN-2 and APH-1aL as in Fig. 1. (c) APH-1aL interacts with immature Nct in the absence of PS and PEN-2. CHAPS-extracted membrane proteins from PS1/2+/+ and PS1/2–/– mouse embryonic fibroblast cells were immunoprecipitated with antibody N1660 (Nct) and analysed by immunoblotting as in Fig. 1.

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Antibodies
  5. cDNA constructs
  6. Cell culture, cell lines, and transfections
  7. Protein analysis
  8. Trypsin-resistance assay
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

γ-Secretase is a high molecular mass complex composed of at least four subunits, namely PS1 or PS2, Nct, APH-1, and PEN-2 (De Strooper 2003), which have been shown to constitute the minimal set of components required for γ-secretase activity of the complex (Edbauer et al. 2003). Besides PS, which constitutes the active site of γ-secretase, little is known about the individual function of the γ-secretase complex components. In addition, little is known about the assembly of a functional γ-secretase complex. Nct may be a key component required for assembly as its expression is not eliminated upon the knock-down of any γ-secretase complex components. Instead it fails to maturate and accumulates as an immature protein within the ER (Edbauer et al. 2002; Kimberly et al. 2002; Lee et al. 2002; Leem et al. 2002; Steiner et al. 2002; Chen et al. 2003; Gu et al. 2003; Herreman et al. 2003). These findings let us to speculate that Nct may represent an initial and first binding partner for sequential assembly of the remaining partners. However, the first binding partner on immature endogenous Nct remained to be identified. During our mutagenesis analysis, we identified rather surprising Nct mutants. These mutants (C248S, E333Q, and G339A) failed to restore γ-secretase function. Moreover, they also failed to restore the levels of PS fragments and PEN-2. Surprisingly, however, APH-1aL levels were stabilized, suggesting that these mutants formed a rather stable association with APH-1aL, which was proven by co-immunoprecipitation analysis. This is in accordance with the model that Nct may be a scaffold for γ-secretase complex assembly and suggests that APH-1 may bind to immature Nct as early as within the ER. To obtain further evidence for a stable Nct/APH-1 interaction we performed experiments in embryonic fibroblast cells derived from PS1/2 double knock-out mice (Herreman et al. 2000). As we and others demonstrated previously (Edbauer et al. 2002; Kimberly et al. 2002; Leem et al. 2002; Chen et al. 2003) Nct fails to maturate in these cells and accumulates as an immature species. Consistent with previous findings we also observed that PEN-2 levels were severely reduced (Steiner et al. 2002; Nyabi et al. 2003) while APH-1aL expression appeared to be rather unaffected (Gu et al. 2003; Nyabi et al. 2003). This is in clear contrast to our findings in Nct knock-down cells. In these cells, both PEN-2 and APH-1aL are strongly reduced (Steiner et al. 2002; Gu et al. 2003; Shirotani et al. 2003). Taken together, these findings may suggest that Nct binds to APH-1aL to form an initial complex as an intermediate during γ-secretase complex assembly. Such a stable Nct/APH-1 assembly intermediate may be supported by our finding that immature Nct and APH-1aL could be co-immunoprecipitated in the absence of PS in PS1/2 double knock-out cells. Interestingly, a potential Nct/APH-1 assembly intermediate has also been observed recently by blue native gel electrophoresis (LaVoie et al. 2003). Additional binding of PS and PEN-2 may be the next step in the assembly of the γ-secretase complex. Finally, following complex formation of the four components, PEN-2 may stimulate PS endoproteolysis as reported previously (Luo et al. 2003; Takasugi et al. 2003). This in turn may initiate transport of the fully assembled complex to its functional sites within the secretory pathway.

Further work will be required to address the question, whether a bona fide Nct/APH-1 assembly intermediate occurs under physiological conditions or whether this interaction rather reflects strong affinities of Nct and APH-1 to each other. However, this question will be difficult to answer as this will require the analysis of γ-secretase complex assembly under nascent conditions. Our finding, that no dose–response relationship to APH-1aL levels was observed in independent cell lines expressing various amounts of wt Nct may indicate that stable assembly intermediates of immature Nct and APH-1 are not formed under conditions of wt complex assembly. On the other hand, complex formation may occur so fast that stable assembly intermediates of immature Nct and APH-1 are difficult to detect.

As demonstrated in this study, binding of PS and PEN-2 to a potential Nct/APH-1 assembly intermediate critically depends on the integrity of the Nct ectodomain as judged from the failure of the C248S, E333Q and G339A mutants to stably associate with PS and PEN-2. Thus, these mutants may either directly affect binding sites of Nct to PS and PEN-2 or they may alter the conformation of Nct such that binding cannot occur. Indeed, the mutants failed to adopt the trypsin-resistant conformation that is observed with wt Nct. The amino acid residues Glu333 and Gly339 are of particular interest because they are part of or are located in close vicinity to the highly conserved DYIGS domain. Interestingly, this domain tolerates the D336/Y337 double mutation but is sensitive to mutation of Gly339 or of the preceding Glu333 thus pointing to an important structure-determining role of the domain by the flanking residues Glu333 and Gly339. Thus, an important future task will be the determination of the crystal structure of the Nct ectodomain to elucidate the molecular basis for the observed effects of these interesting mutants.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Antibodies
  5. cDNA constructs
  6. Cell culture, cell lines, and transfections
  7. Protein analysis
  8. Trypsin-resistance assay
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 596, Molecular Mechanisms of Neurodegeneration) and the European Community. We thank Dr R. Agami for the pSUPER vector, Dr B. De Strooper for PS1/2 deficient mouse embryonic fibroblast cells, Dr R. Nixon for the monoclonal antibody PS1N, and Drs G. Yu, Y. Gu and P. St George-Hyslop for Nct cDNA constructs and the APH-1aL antibody.

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Antibodies
  5. cDNA constructs
  6. Cell culture, cell lines, and transfections
  7. Protein analysis
  8. Trypsin-resistance assay
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References
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