Pen-2 and Presenilin are Sufficient to Catalyze Notch Processing

Cell culture

Mouse embryonic fibroblast (MEF) knockout cells Aph-1^–/– [11] and NCT^–/– [12] were provided by Dr. Tong Li from John Hopkins University. Pen-2^–/– [8], PS1^–/– [13], PS2^–/– [14], PS1/2^–/– [15], and wild type (WT) MEF cells were provided by Dr. Bart De Strooper from the Center of Human Genetics (Belgium). Wt-7 cells, which express both human PS1 and Swedish mutant AβPP, was provided by Drs. Sangram S. Sisodia and Seong-Hun Kim from the University of Chicago. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Walkersville, MA, USA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine (Lonza), 100  units/mL penicillin (Lonza), and 100 μg/mL streptomycin (Lonza).

Inhibitors and reagents–γ-secretase inhibitors Compound E and L685 were purchased from EMD Millipore (Billerica, MA, USA). Complete protease inhibitor cocktail tablets were purchased from Roche Applied Science (Indianapolis, IN, USA). Lipofectamine LTX and plus reagent and Lipofectamine RNAiMAX reagent were purchased from Invitrogen (Carlsbad, CA, USA).

Antibodies–Anti-PS1C antibodies (#5643 and #3622) and NICD-specific antibody (#4147) were purchased from Cell Signaling (Danvers, MA). Polycolonal antibody C15 was raised against the last 15 amino acids at the C terminal of AβPP [16]. Monoclonal antibody 6E10 and Polyclonal antibody anti-PEN-2N were from Covance (Emeryville, CA). Anti-myc antibody (9E10) was purchased from Santa Cruz (Dallas, TX, USA). Anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was from EMD Millipore (Billerica, MA). Polycolonal NCT antibody N1660 was purchased from Sigma-Aldrich(St. Louis, MO). Anti-PS1N was raised against a peptide corresponding to residues 27–50 of humanPS1 [17].

Plasmids

Plasmid expressing the ectodomain truncated and myc-tagged Notch molecule (NotchΔE) containing the murine Notch-1 leader peptide (1–23 aa) [18] was kindly provided by Raphael Kopan (Washington University) and Dr. Masayasu Okochi (Osaka University, Japan). The plasmid AβPPsw, which expresses a C-terminal myc-tagged Swedish mutant AβPP (AβPPsw) [19], was kindly provided by Dr. Gopal Thinakaran (University of Chicago). The plasmids, expressing the PS1N terminal (1–292aa), PS1C terminals: PS1C₂₉₃ (293–467aa), PS1C₂₉₆ (296–467aa), PS1C₂₉₉ (299–467aa), PS1C₃₃₄(334–467aa), and PS1C₃₄₆ (346–467aa), were constructed in our laboratory and confirmed by sequencing. PS1 mutants PS1_D257A, PS1_D385A, and PS1_D257,385A expressing plasmids were constructed as previously described [20].

siRNA treatment

NCT specific siRNAs and control siRNA were purchased from Qiagen (Valencia, CA, USA). Cells were treated with siRNA twice every two days using Lipofectamine RNAiMAX reagent following manufacturer’s instructions. After four days of treatment and confirmation of NCT-knockdown, cells were transfected with NotchΔE or lacZ for another 24 h followed by western blotting analysis.

Immunoprecipitation and western blot analysis

Twenty-four hours after transfection, cells were collected with 1% Chapso buffer (20 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA and cocktail). The collected samples were then immunoprecipitated with appropriate antibodies and Protein A conjugated beads overnight at 4°C followed by western blot analysis as described previously [9]. Protein A sepharose was purchased from Pharmacia Biotech (Piscataway, NJ). Chapso was purchased from Amresco (Solon, OH).

Pen-2 is required for the processing of both AβPP and Notch

To determine the roles of each components of γ-secretase complex in the γ-secretase activity, knockout cells with deletion of one of these components were cultured in the presence or absence of γ-secretase inhibitor L685–458 (L685). After 24 h of culture, cell lysates were probed with C15, which is specific to the C-terminal fragment of AβPP [16]. As shown in Fig. 1A, in the absence of inhibitor, the level of CTFα, which is produced by α-secretase from AβPP, dramatically reduced in Aph-1 knockout cells (Aph-1^–/–) (lane 1), PS1^–/– cells (lane 7), PS2^–/– cells (lane 9), as well as in wild type cells (lanes 13). When these cells were cultured in the presence of inhibitor, significant amount of CTFα was accumulated (lanes 2, 8, 10, and 14). However, regardless of the presence or absence of inhibitor, a high-level accumulation of CTFα was detected in nicastrin knockout (NCT^–/–), Pen-2 knockout (Pen-2^–/–), and PS1 and PS2 double knockout (PS1/2^–/–) cells (lanes 3, 4, 5, 6, 11, and 12). These results indicate that NCT and Pen-2 are essential for γ-secretase catalyzed CTFα turnover, but Aph-1 is dispensable. As shown in the top panel of Fig. 1A, relatively uniform expression levels of full-length AβPP (fAβPP) were detected through all of the cell lines. It was noted that the level of fAβPP in NCT^–/– cells is slightly lower than those in other cells, suggesting a relatively quick turnover of fAβPP in NCT-deficient cells either by random degradation or by α- and/or β-secretase, a question awaiting further investigation. Next, we examined the effect of knockout of these components on the processing of Notch. Cells were transfected with plasmid expressing NotchΔE, a ectodomain truncated form of Notch. As shown in Fig. 1B, in the absence of inhibitor, a significant amount of NICD, which is produced from NotchΔE by γ-secretase activity, was detected in Aph-1^–/– cells (lane 1), NCT^–/– cells (lane 3), PS1^–/– cells (lane 7), and PS2^–/– cells (lane 9) as well as in wild type cells (lane 13). When these cells were cultured in the presence of inhibitor, the NICD was reduced with a concomitant increase in the level of unprocessed NotchΔE (lanes 2, 4, 8, 10, and 14). Interestingly, in the Pen-2^–/– cells (lanes 5 and 6) as well as PS1/2^–/– cells (lanes 11 and 12), no NICD was detected regardless of the presence or absence of inhibitor. These results suggest that, unlike Aph-1 and NCT, Pen-2 is crucial for γ-secretase catalyzed processing of Notch. It was noted that, in the presence of the inhibitor, the levels of unprocessed NotchΔE accumulated in Aph-1^–/–, PS2^–/–, and PS1^–/– cells (lanes 2, 8, and 10) are lower than those expected and this may be the result of random degradation of the unprocessed NotchΔE in these cells deficient in these γ-secretase components.

Pen-2 together with presenilin can form a functionally active γ-secretase for the processing of Notch

As reported in our previous study [21], data presented in Fig. 1A and B revealed very interesting findings that Aph-1 is dispensable for processing of both AβPP and Notch, that NCT is required for AβPP processing but not for Notch processing, and that Pen-2 is absolutely required for the processing of both AβPP and Notch. These findings suggest that there is a different requirement for NCT in processing of Notch and AβPP. Specifically, the finding that neither Aph-1 nor NCT was absolutely required for Notch processing when these genes were individually inactivated prompted us to determine the effect of inactivation of both Aph-1 and NCT genes simultaneously on the processing of Notch. As shown Fig. 1C, all three NCT-specific siRNAs efficiently blocked the expression of NCT (third panel, lanes 3–5) in Aph-1^–/– cells. Intriguingly, the level of NICD was not changed upon knockdown of NCT in Aph-1^–/– cells (compare lanes 3–5 with lanes 1–2).These data strongly indicate that Pen-2 is absolutely essential and sufficient for supporting presenilin-catalyzed Notch processing.

Ectopically expressed PS1N and PS1C are stable, but unable to restore γ-secretase activity in Pen-2^–/– cells

Previous studies have revealed that Pen-2 plays roles in efficient proteolytic processing of presenilin and stabilization of the resulting N-terminal andC-terminal fragments of presenilin [9, 10]. Thus, to determine whether these roles account for the absolutely requirement of Pen-2 for presenilin-catalyzed Notch processing, we first examined the stability and the enzymatic activity of the N-terminal fragment (PS1N) and the C-terminal fragment (PS1C) of PS1 in Pen-2 knockout cells (Pen-2^–/–). Since PS1 undergoes heterogeneous endoproteolysis between Thr₂₉₁ and Ala₂₉₉ and can also be processed by caspase activity to produce PS1Cs with different length [22–25], we constructed a series of plasmids (namely PS1C₂₉₃, PS1C₂₉₆, PS1C₂₉₉, PS1C₃₃₄, and PS1C₃₄₆) expressing different PS1Cs starting from amino acid 293, 296, 299, 334, and 346 to the very end of C terminal and a plasmid PS1N expressing the N-terminal fragment of PS1 from amino acid 1 to 292. Pen-2^–/– cells were co-transfected with NotchΔE along with a combination of PS1N and one the PS1Cs of different lengths. As shown in Fig. 2A, all recombinant proteins were expressed at significant levels. Specifically, the high levels of PS1N and PS1C detected in these cells indicate that the ectopically expressed PS1N and PS1C are stable in Pen-2^–/– cells. It was noted that in the PS1wt-expressing cells the PS1N and PS1C produced by normal endoproteolytic processing were detected at relatively low levels and the most abundant PS1C detected is the one equivalent to the PS1C produced by caspase activity (top panel, lane 4). This may be due to the inefficient endoproteolytic processing of PS1 and the instability of the resulting PS1N and PS1C in the absence of Pen-2 as reported by previous study [9]. As a control, Wt-7 cells were also transfected with NotchΔE. As shown in fourth panel, in Wt-7 cells, a significant amount of NICD was detected (lane 10). However, no NICD was detected in Pen-2^–/– cells co-transfected either with a combination of PS1N and PS1C (third panel, lanes 5–9) or with PS1wt (lane 4). The fact that NICD was not detected in Pen-2^–/– cells expressing significant amounts of PS1C and PS1N strongly suggest that in addition to enhancing PS1 endoproteolytic processing and stabilization of the resulting PS1N and PS1C fragments, Pen-2 must play a more crucial role in in catalytic activity of γ-secretase.

Pen-2 is not required for the heterodimer formation and stabilization of PS1N and PS1C

The presenilin NTF/CTF heterodimers are considered to constitute the active site in the γ-secretase complex [26–28]. A study has also suggested that Pen-2 may be required for the stabilization of PS1N/PS1C heterodimer [29]. Thus, we examined the effect of knockout of Pen-2 on the formation of the PS1N/PS1C heterodimer. As shown in the upper panel of Fig. 2B, when the cells were transfected with PS1wt, a band corresponding to PS1C₂₉₃ was co-immunoprecipitated with PS1N by anti-PS1N antibody (lane 2). Similarly, significant amount of PS1Cs were co-immunoprecipitated by anti-PS1N antibody from cells expressing PS1N and various PS1Cs including the PS1Cs produced by caspase activity (lanes 3–7). The similar co-immunoprecipitation results were also observed in wild type MEF cells (lane 8) and Wt-7 cells(lane 9). These results indicate that Pen-2 is not required for the formation and stabilization of PS1N/PS1C heterodimer.

Pen-2 is required for Notch binding to PS1

Next, we determined the effect of knockout of Pen-2 on the binding of Notch to γ-secretase. Cells were transfected with NotchΔE alone with either non-related protein LacZ or plasmids expressing PS1 variants. As shown in Fig. 2C, when the cell lysates were immunoprecipitated with anti-Myc antibody, high levels of NotchΔE was detected in all transfectants (top panel, lanes 3, 6, 9, 12, and 15). When the cell lysates were subjected to co-immunoprecipitation using anti-Pen-2 antibody, as shown in lane 10 of top panel, NotchΔE was readily co-immunoprecipitated with Pen-2 in wild type cells. However, anti-PS1C antibody pulled down hardly any NotchΔE in the absence of γ-secretase inhibitor (lane 11). This is likely due to the activity of γ-secretase that processes NotchΔE into NICD, which is no longer associated with PS1. Indeed, NotchΔE was pulled down by anti-PS1C antibody in wild type cells in the presence of γ-secretase inhibitor compound E (lane 14, top panel). It was also noted that, in the presence of compound E, the level of NotchΔE pulled down by anti-Pen-2 antibody was also increased and higher than that pulled down by anti-PS1C antibody (compare lane 13 with lane 10 and lane 13 with 14), suggesting that the association of Notch with Pen- 2 is stronger than that with PS1. This notion was strongly supported by the results that NotchΔE was readily co-immunoprecipitated with Pen-2 in PS1/2^–/– cells (lane 1, top panel), indicating that Pen-2 is capable of interacting with NotchΔE in the absence of presenilin. However, in Pen-2^–/– cells, anti-PS1C antibody did not bring down any NotchΔE (lane 5, top panel), even in the presence of ectopically expressed PS1N and PS1C. It was also noted that when the PS1/2^–/– cells were transfected with PS1N and PS1C, not only anti-PS1C antibody but also anti-Pen-2 antibody brought down significantly more NotchΔE than that in cells without overexpression of PS1 (compare lane 8 with lane 11 and lane 7 with lane 10). These results suggest that PS1 also enhances the interaction between Pen-2 and NotchΔE. In addition, this set of experiments further confirmed that, similar to PS1/2^–/– cells, Notch is not processed in the absence of Pen-2— even in the presence of overexpressed PS1 (lane 6, second panel). It was also noted that NICD was immunoprecipitated by anti-Myc antibody (lanes 9 and 12, second panel), but not by anti-Pen-2 antibody (lanes 7 and 10, second panel) nor by anti-PS1C antibody (lanes 8 and 11), suggesting that, upon formation, NICD was released from the γ-secretase complex. In addition, in wild type cells, PS1C was co-immunoprecipitated with Pen-2 by anti-Pen-2 antibody (lanes 10 and 13, third and fourth panels) vice versa (lanes 11 and 14, third and fourth panels). Note that the bands detected in lanes 1, 4, 7, 10, and 13 in second and third panels are non-specific bands corresponding to heavy chain and light chain of anti-Pen-2 antibody, respectively.