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Dominant negative effect of the loss-of-function γ-secretase mutants on the wild-type enzyme through heterooligomerization

  1. Yigong Shia,b,c,d,2
  1. aBeijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing 100084, China;
  2. bTsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing 100084, China;
  3. cCenter for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China;
  4. dInstitute of Biology, Westlake Institute for Advanced Study, Xihu District, Hangzhou 310064, Zhejiang Province, China
  1. Contributed by Yigong Shi, September 12, 2017 (sent for review August 2, 2017; reviewed by Yue-Ming Li, Jie Shen, and Gang Yu)

  1. Fig. S1.

    The dominant negative effect of the catalytically inactive γ-secretase-DD (PS1-D257A, D385A) over the WT γ-secretase in terms of the production of Aβ40 and Aβ42. (A) The proteolytic activities of WT γ-secretase and six representative mutants (18). Shown here are the combined production of Aβ40 and Aβ42. The proteolytic activity of WT γ-secretase is normalized as 1. (B) The dominant negative effect of the catalytic mutant γ-secretase-DD over WT γ-secretase in terms of the production of Aβ40. (C) The dominant negative effect of the catalytic mutant γ-secretase-DD over WT γ-secretase in terms of the production of Aβ42. (D) The ratio of Aβ42 over Aβ40 remains largely the same. The Aβ42/Aβ40 ratio of WT γ-secretase alone is normalized as 1. **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.

  2. Fig. 2.

    The loss-of-function γ-secretase mutants, each containing an AD-derived mutation in PS1, inhibit the production of Aβ40 and Aβ42 by WT γ-secretase. (A) Dominant negative effect of the γ-secretase mutant (PS1-Y115H) on WT γ-secretase. The concentration of WT γ-secretase is 16 nM in A–E. The total amount of Aβ40 and Aβ42 produced by WT γ-secretase alone is normalized as 1 in all panels. Each experiment was repeated three times, with the SD shown. (B) Dominant negative effect of the γ-secretase mutant (PS1-L166P) on WT γ-secretase. (C) Dominant negative effect of the γ-secretase mutant (PS1-C410Y) on WT γ-secretase. (D) Dominant negative effect of the γ-secretase mutant (PS1-L435F) on WT γ-secretase. (E) The γ-secretase mutant (PS1-S365A), which has a proteolytic activity comparable to that of the WT γ-secretase, exhibits no dominant negative effect. In fact, the proteolytic activity increases with increasing amounts of the mutant γ-secretase. (F) A summary of the dominant negative effect by five γ-secretase mutants. The concentrations of WT and mutant γ-secretases are 16 and 48 nM, respectively, in each case.

  3. Fig. 3.

    γ-secretase molecules interact with each other in the presence of the detergent CHAPSO. (A) WT γ-secretase interacts with the catalytic mutant γ-secretase (PS1-D257A, D385A). All pull-down experiments described in this figure were performed using purified recombinant proteins. In the experiments described in A and B, the WT and mutant γ-secretases are differentially tagged and incubated together. Immunoprecipitation using one specific antibody was followed by Western blots using another specific antibody. (B) WT γ-secretase interacts with each of the three mutant γ-secretase proteins (PS1-Y115H, C410Y, and ΔE9). (C) WT or catalytic mutant γ-secretase forms oligomers. In these experiments, the WT (or catalytic mutant) γ-secretase proteins are differentially tagged. The pull-down experiments were performed to assess the WT–WT or mutant–mutant γ-secretase interactions, with WT–mutant interactions as the control. A similar pull-down efficiency is observed in all combinations.

  4. Fig. S2.

    γ-secretase molecules interact with each other in the presence of the detergent CHAPSO. (A) WT γ-secretase interacts with the catalytic mutant γ-secretase-DD (PS1-D257A, D385A). All pull-down experiments described in this figure were performed using purified recombinant proteins. The WT and mutant γ-secretases are differentially tagged and incubated together. Immunoprecipitation using one specific antibody was followed by Western blots using another specific antibody. Compared with the experiments described in Fig. 3, the tags were swapped for the WT and mutant γ-secretases. (B) WT γ-secretase interacts with each of the three mutant γ-secretase proteins (PS1-Y115H, C410Y, and ΔE9).

  5. Fig. 4.

    The interactions among γ-secretase molecules are strictly dependent on the choice of detergents and correlate with the proteolytic activity. (A) WT γ-secretase exhibits robust proteolytic activity in the reaction buffer containing CHAPSO, but not digitonin or amphipol A8-35. WT γ-secretase was purified in three different detergents: CHAPSO, digitonin, and amphipol A8-35. Then the proteolytic activity assays were performed in three different buffers. Regardless of the original detergent used in purification, WT γ-secretase is highly active, as long as the reaction buffer contains the detergent CHAPSO. Each experiment was repeated three times, with the SD shown. (B) γ-secretase containing catalytic mutations and WT γ-secretase cannot pull down each other in digitonin buffer. (C) γ-secretase containing catalytic mutations and WT γ-secretase in amphipol A8-35 cannot pull down each other.

  6. Fig. S3.

    The interactions between WT and catalytic mutant γ-secretases are strictly dependent on the choice of detergents. (A) The WT and catalytic mutant γ-secretases cannot pull down each other in the digitonin buffer. Compared with the experiments described in Fig. 4, the tags were swapped for the WT and mutant γ-secretases. (B) The WT and catalytic mutant γ-secretases cannot pull down each other in the reaction buffer containing amphipol A8-35.

  7. Fig. 5.

    Distinct oligomerization states of γ-secretase in different detergents and γ-secretase form a suspicious dimer after GraFix. (AC) Representatives of the analytical ultracentrifugation results of γ-secretase in different detergents as indicated. (D) The negative staining image of γ-secretase in digitonin. (E) Negative staining image of γ-secretase in CHAPSO. (F) Negative staining image of GraFixed γ-secretase. (G) Representative negative staining 2D classification of GraFixed γ-secretase.

  8. Fig. S4.

    Results of the cell-based cleavage assays are consistent with a dominant negative effect by mutant γ-secretases. (A) Location of the amino acids targeted for mutations in the PS1 structure. Except for Asp333 in the disordered loop between TM6 and TM7, the other nine amino acids targeted for mutations are distributed throughout all nine TMs of PS1. (B) Determination of the amount of PS1-expressing plasmid for transfection into 1 mL HeLa cells. The combined production of Aβ40 and Aβ42 was monitored. Within the range of 7.8–62.5 ng of the transfected PS1 plasmid, the total amount of Aβ40 and Aβ42 is roughly linearly proportional to the amount of transfected PS1-expressing plasmid under the condition of fixed C99-expressing plasmid (500 ng). (C) The expression levels of the WT and mutant PS1 proteins in HeLa cells. Plasmids that overexpress the WT and mutant PS1 proteins were transfected into PSEN1/PSEN2 double-knockout HeLa cells. Western blots were performed on these cells using an anti-PS1 monoclonal antibody. The total amount of PS1 is similar for these different transfections. The label “1/2” indicates one-half of the full amount (62.5 ng) of that particular plasmid for transfection into 1 mL HeLa cells. For example, “1/2 WT + 1/2 DD” means 31.25 ng of the plasmid for WT PS1 plus 31.25 ng of the plasmid for the catalytic mutant PS1-DD. (D) The combined production of Aβ40 and Aβ42 was detected from medium after transfection of plasmids carrying PS1(62.5 ng as full amount) and the substrate APP-C99 (500 ng). Coexpression of 1/2 loss-of-function PS1 mutants with 1/2 WT PS1 results in compromised activities compared with 1/2 WT alone, indicating a dominant negative effect. This effect applies to all four PS1 mutants: D257A/D385A, G384A, C410Y, and L435F. (E) The production of Aβ40 in the cell-based assay. (F) The production of Aβ42 in the cell-based assay.

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