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. 2012;7(3):e32293.
doi: 10.1371/journal.pone.0032293. Epub 2012 Mar 30.

Modulation of γ-Secretase Activity by Multiple Enzyme-Substrate Interactions: Implications in Pathogenesis of Alzheimer's Disease

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Free PMC article

Modulation of γ-Secretase Activity by Multiple Enzyme-Substrate Interactions: Implications in Pathogenesis of Alzheimer's Disease

Zeljko M Svedružić et al. PLoS One. .
Free PMC article

Abstract

Background: We describe molecular processes that can facilitate pathogenesis of Alzheimer's disease (AD) by analyzing the catalytic cycle of a membrane-imbedded protease γ-secretase, from the initial interaction with its C99 substrate to the final release of toxic Aβ peptides.

Results: The C-terminal AICD fragment is cleaved first in a pre-steady-state burst. The lowest Aβ42/Aβ40 ratio is observed in pre-steady-state when Aβ40 is the dominant product. Aβ42 is produced after Aβ40, and therefore Aβ42 is not a precursor for Aβ40. The longer more hydrophobic Aβ products gradually accumulate with multiple catalytic turnovers as a result of interrupted catalytic cycles. Saturation of γ-secretase with its C99 substrate leads to 30% decrease in Aβ40 with concomitant increase in the longer Aβ products and Aβ42/Aβ40 ratio. To different degree the same changes in Aβ products can be observed with two mutations that lead to an early onset of AD, ΔE9 and G384A. Four different lines of evidence show that γ-secretase can bind and cleave multiple substrate molecules in one catalytic turnover. Consequently depending on its concentration, NotchΔE substrate can activate or inhibit γ-secretase activity on C99 substrate. Multiple C99 molecules bound to γ-secretase can affect processive cleavages of the nascent Aβ catalytic intermediates and facilitate their premature release as the toxic membrane-imbedded Aβ-bundles.

Conclusions: Gradual saturation of γ-secretase with its substrate can be the pathogenic process in different alleged causes of AD. Thus, competitive inhibitors of γ-secretase offer the best chance for a successful therapy, while the noncompetitive inhibitors could even facilitate development of the disease by inducing enzyme saturation at otherwise sub-saturating substrate. Membrane-imbedded Aβ-bundles generated by γ-secretase could be neurotoxic and thus crucial for our understanding of the amyloid hypothesis and AD pathogenesis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Different phases in γ-secretase reaction can be separated in time.
The reactions were prepared using CHAPSO enriched γ-secretase membranes (total protein 0.25 mg/ml) and saturating concentration of C99 substrate (3.0 µM). (A–B). Early time points and the pre-steady-state for AICD, Aβ 1–40 and Aβ 1–42 production (panel B is zoom-in on panel A). The best-fit profile for AICD production was calculated using the equation for pre-steady-state burst (eqn. 1, Table 1), while the best-fit profiles for Aβ 1–40 and Aβ 1–42 production were calculated using the equation for enzyme hysteresis (eqn. 2, Table 1). (C) The time profiles Aβ 1–40 and Aβ 1–42 from figure 1 were used to calculate the changes in Aβ42/Aβ40 ratio as a function of the reaction time (Aβ42/Aβ40 ratio is shown, rather than Aβ40/Aβ42 ratio, in an attempt to follow standards in the literature).
Figure 2
Figure 2. Urea gels show Aβ 1-x products in different phases of γ-secretase reaction.
The reactions were prepared using CHAPSO enriched γ-secretase membranes (total protein 0.25 mg/ml), and saturating concentration of C99 substrate (3.0 µM). The lanes “Aβ std 1-x” represents synthetic peptides as mobility standards. To facilitate detection of the early data points ( A ) the reaction volume was twenty fold bigger than usual, and the resulting 1-x Aβ products were concentrated about twenty-fold by immunoprecipitation using protein G beads and polyclonal antibodies specific for the first 5 amino acids. It is necessary to mention that pre-incubation of the assay mix for several hours prior to the start of reaction (i.e. addition of C99 substrate) does not affect the relative distribution of different Aβ products. Therefore, the observed changes are not due to enzyme denaturation during the course of the reaction.
Figure 3
Figure 3. Michaelis-Menten profiles for AICD, Aβ 1–40 and Aβ 1–42 in presence of DAPT.
CHAPSO enriched γ-secretase membranes were used to measure Michaelis-Menten profiles for total AICD production in presence of 0 nM (•), 70 nM (+) and 150 nM (O) of DAPT. Michaelis-Menten profiles for Aβ 1–40 and Aβ 1–42 production were measured in presence of 0 nM (•), 100 nM (+) and 200 nM (O) of DAPT. All profiles have been analyzed using nonlinear regression and the eqn. 4 (methods). The corresponding best fit values are summarized in Table 2. The gel strips show different concentrations of the C99 substrate and the corresponding AICD products. Alternating in-between are the parallel control reactions in which γ-secretase was inhibited by a mix of 10 µM of DAPT and LY-411,575 , . AICD was measured using antiflag M2 antibodies (as shown in the gel strip). Aβ 1–40, and Aβ 1–42 were measured using AlphaScreen® as described in methods section.
Figure 4
Figure 4. Changes in Aβ products caused by gradual saturation of γ-secretase.
Saturation of γ-secretase with its C99 substrate leads to decrease in Aβ40 production with concomitant increase in production of the longer more hydrophobic Aβ peptides and Aβ42/Aβ40 ratio. (A) The saturation profiles from Fig. 3 were used to calculate the ratio between Aβ 1–40 (•) and Aβ 1–42 (O) production and the total AICD production. The ratio curves were calculated using the saturation profiles from Fig. 3 in the absence of DAPT. ( B ) Urea gels were used to analyze the relative distribution of different Aβ 1-x fragments at half-saturating (0.3 µM) and saturating (3.0 µM) concentrations of C99 substrate. The lane “Aβ std 1-x” represents synthetic peptides as mobility standards, the lane “inhibitor” represents parallel control reaction in the presence of 10 µM of γ-secretase inhibitors DAPT and LY-411,575 , . ( C ) The relative intensity of each Aβ 1-x peak is shown as a percent of the total sum of all Aβ peaks in the corresponding lane. The intensity of different Aβ 1-x products was quantified by transforming the individual bands into a series of peaks using the “ribbon option” in program ImmageQuant 5.0. The resulting peaks and the corresponding baselines were quantified using the “peak-fit” option in MicroCal Origin 7.0 program.
Figure 5
Figure 5. Oligomerization of C99 substrate.
C99 dimerization/oligomerization was measured using aliquots of C99 substrate that had high activity with γ-secretase in CHAPSO enriched membranes. Oligomerization between C99 molecules was measured using AlphaScreen® technology by coupling both the donor-beads, and the acceptor-beads, to 3D6 antibody (right panel). Increasing concentration of C99 substrate was incubated with 10 nM of 3D6 monoclonal antibodies coupled to either donor or acceptor-beads. Since one epitope can bind only one antibody, the acceptor and the donor beads can come to proximity and give the AlphaScreen® signal only if C99 dimerization/oligomerization brings the epitopes together (right panel). A nonlinear regression and the equation 5 (methods) were used to calculate an apparent dissociation constant, Kd = 33±2 nM .
Figure 6
Figure 6. NotchΔE substrate can activate γ-secretase activity on C99 substrate.
γ-Secretase activity in CHAPSO enriched membranes was measured using half-saturating C99 substrate ([C99] = 0.45 µM, fresh after purification) in the presence of increasing concentration of NotchΔE substrate (•), and in identical control assays without NotchΔE substrate (O). The AICD production was measured using 125-I labeled C99 substrate and autoradiography as shown on the gel strips (125-I assay was used instead of western blot since both substrates were purified using antiflag M2 epitopes, see methods). Different interactions between γ-secretase and its C99 (black helix) or NotchΔE (green helix) substrates can be illustrated using a model mechanism. C99 substrate can be shown as a transmembrane helix , while γ-secretase can be shown as a bowl-shaped membrane-imbedded complex . The underlined numbers connect the different complexes with the corresponding activity range on the graph. In a simple scenario, of one enzyme binding one substrate, NotchΔE and C99 substrates could be only competitive inhibitors . We find that NotchΔE substrate can activate γ-secretase reaction on C99 substrate (1). Such scenario can happen only if γ-secretase can bind both substrates at the same time (2). NotchΔE substrate shows competition with C99 substrate only when its concentration is several folds higher than C99 concentration (3). Extrapolation of the presented profile shows that close to 10 µM of NotchΔE substrate would be needed for a full inhibition (4).
Figure 7
Figure 7. γ-Secretase is not affected by Aβ peptides present in free solution.
(A) The lanes labeled as “free reaction” represent Aβ products after 4 hours of routine γ-secretase reaction at half saturating C99 substrate ([C99] = 0.45 µM). The lanes labeled as “+10 nM Aβ 1–42” and “+10 nM Aβ 1–40” represent “free reaction” premixed with synthetic Aβ 1–42 or Aβ 1–40 in a concentration equivalent that corresponds to 4 hours of free reaction. The lanes labeled “+21F12” and “+2G6” represent “free reaction” that was premixed with antibodies specific for Aβ42 or Aβ40 respectively. Both 2G6 and 21F12 antibodies bind the matching Aβ peptides very efficiently as indicated by a complete removal of the corresponding Aβ bands in the reactions with protein G beads (samples labeled as “21F12 and prot. G” and “2G6 and prot. G”). The lane “Aβ std 1-x” represents synthetic peptides as mobility standards. (B) AICD production was measured at half-saturating C99 substrate (0.45 µM) in assays that were premixed with increasing concentrations of synthetic Aβ 1–38, Aβ 1–40, Aβ 1–42, or Aβ 1–44.
Figure 8
Figure 8. AICD and Aβ production by WT presenilin 1 and two FAD mutants.
CHAPSO enriched γ-secretase membranes carrying WT presenilin 1 or FAD mutants ΔE9 and G384A have been prepared and analyzed in parallel with all conditions identical. ( A ) Michaelis-Menten profiles for total AICD production (i.e. the turnover rates [37]) were measured in parallel using anti-flag αM2 antibodies as shown in Fig 3. ( B ) urea gels show the relative distribution of different Aβ 1-x products at the sub-saturating and saturating substrate concentrations (5 hour reactions). The lane “Aβ std 1-x” represents synthetic peptides as mobility standards, the lane “inhibitor” represents a parallel control reaction in the presence of 10 µM of γ-secretase inhibitors DAPT and LY-411,575 , ( C ) The relative intensity of each Aβ 1-x peak is shown as a percent of the total sum of all Aβ peaks in the corresponding lane. The intensity of different Aβ 1-x products was quantified by transforming the individual bands into a series of peaks using the “ribbon option” in program ImmageQuant 5.0. The resulting peaks and the corresponding baselines were quantified using the “peak-fit” option in MicroCal Origin 7.0 program.
Figure 9
Figure 9. Inhibition of WT presenilin 1 and FAD mutants by DAPT and L-685,458.
CHAPSO enriched γ-secretase membranes carrying WT presenilin 1 or FAD mutations ΔE9 and G384A have been prepared and analyzed in parallel with all conditions identical. The dose response curves for DAPT and L-685,458 were measured by following total AICD production using western-blots with αM2 antiflag antibody as shown in Fig. 3. The results were analyzed using nonlinear regression and the equation 3 (methods). The best fit values and the corresponding statistics are given in Table 3.
Figure 10
Figure 10
Steps in the catalytic cycle of γ-secretase. The model illustrates the basic biophysical principles of processive cleavages and intramembrane proteolysis , , , , , . C99 substrate can be shown as a transmembrane helix , while γ-secretase can be shown as a bowl-shaped membrane-imbedded complex with its active site aspartates in the central aqueous cavity , , . The initial AICD cleavage (Fig. 1) takes place between amino acids 48–49 or 49–50 , just under the membrane surface , in a dynamic section that has a tendency to destabilize the transmembrane helix ((C1->C4), [58]). The result is a soluble AICD fragment, and a hydrophobic Aβ fragment with its negatively charged carboxyl-terminal trapped below the membrane surface (C3->C4). Thus, the negatively charged carboxyl-terminal is in an energy gap that is forcing it to the interface between the hydrophobic enzyme core and the hydrophilic central aqueous cavity. The opposing force comes from the hydrogen bonds that tend to stabilize the transmembrane helix (C4). The Aβ peptides have a highly dynamic structure that can vary from α-helix to random-coil –, . Such structural changes can drag small parts of the hydrophobic Aβ peptides to the active site aspartates following the negatively charged carboxyl-terminus in the central aqueous cavity ((C4->C7), [11]). Thus, the whole process can be driven by entropy and/or by repulsive forces between negative charges on the active site aspartates and the carboxyl-terminal on the nascent Aβ –, . There is no need for active use of cell's energy. The result is a sequence of processive cleavages of hydrophobic tri-peptides that does not require a full exposure of the hydrophobic substrate to the aqueous catalytic site . The initial cleavage at 49–50 site leads to Aβ 49–46–43–40 sequence, while the initial cleavage at 48–49 site leads to Aβ 48–45–42–38 sequence , , , . It is very important to realize that the most frequent end-products Aβ 1–40 and Aβ 1–42 have more than a half of the original hydrophobic transmembrane helix of C99 (C6->C7). Such products are highly unlikely to spontaneously dissociate from the hydrophobic γ-secretase to the hydrophilic extracellular space (C7c). Furthermore, the peptides are too short to form a transmembrane helix (C7a) , while the fully extended structures (C7b) can not be stabile due to unsatisfied hydrogen bonds in the peptide backbone . For the same reasons the nascent Aβ-peptides (C1->C6) can not be spontaneously released from γ-secretase. The hydrophobic Aβ products can dissociate from γ-secretase only by interacting with a carrier protein, or by forming an Aβ bundle as in Fig. 11. The carrier protein is expected to facilitate catalytic rates since dissociation of Aβ products is the rate-limiting step (Fig. 1, and Fig. S1). Thus, possible candidates for the carrier protein can be the proteins identified by He and coauthors , apo-lipoprotein E , PrP C , or some other surface proteins .
Figure 11
Figure 11. Multiple C99 molecules bound to γ-secretase can facilitate the pathogenesis.
Multiple C99 molecules bound to γ-secretase can affect the catalytic mechanism and contribute to the neurotoxic events. Multiple C99 molecules bound to the enzyme (1) could interact just as free C99 molecules –. Such interactions can influence the initial AICD cleavage and thus control the difference between Aβ 49–46–43–40 or Aβ 48–45–42 cleavage paths (Fig. 10). If multiple C99 molecules are cleaved in parallel, the result will be a bundle of nascent Aβ peptides (3), or even a mixed bundle of C99 and nascent Aβ peptides (2). All of those interactions can be affected by the same structural forces that control interactions between Aβ peptides in free solution. Thus, there could be a preferred number of peptides in the bundle , , and a preferred ratio between Aβ40, Aβ42, and the longer Aβ peptides . Any of those can affect dynamic structural changes that control the processive cleavages, and ultimately the type of Aβ products (Fig. 10). Packed together the nascent Aβ peptides can undergo a series of structural changes so that their β-genic amino acids (Thr, Val, Ile) can initiate formation of extended β-sheet bundles (3->4) –, , . This can drive transition from the α-helix structure of C99 to the β-sheet structure of Aβ oligomers –, , . The whole process can be chaperoned and accelerated by the enclosure within the enzyme structure. Some functional and evolutional links have been observed between chaperones and rhomboid intramembrane proteases , . Unlike single amyloid peptides (Fig. 10), the hydrophobic β-sheet bundles can be easily released into the lipid bilayer (5–>6). The bundles can be stabilized by hydrogen bonding between the peptides' backbones so that their hydrophobic amino acids can face the lipid bilayer . The released β-sheet bundles can accumulate to toxic levels by causing disruption of membrane integrity (i.e. fluidity, lipid rafts and ion gradients [51], [97]). Thus, the neurotoxic processes can start directly in the membrane where toxic amyloid peptides are produced, rather than in the extracellular space as it was suggested in the original amyloid hypothesis and its subsequent derivatives . Extracellular amyloid fibrils can be the end result of chronic toxic overload and the final membrane breakdown (7) .

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