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Modulation of γ-secretase activity by multiple enzyme-substrate interactions: implications in pathogenesis of Alzheimer's disease.

Svedružić ZM, Popović K, Smoljan I, Sendula-Jengić V - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Biochemistry, Faculty of Medicine, University of Rijeka, Rab, Croatia. zsvedruz@biol.pmf.hr

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.

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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.
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pone-0032293-g002: 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.

Mentions: We further analyzed the early stage of the reaction using urea gels (Fig. 2 A–B) that can separate Aβ 1–40 and Aβ 1–42 from the other Aβ 1-x products (the urea gels are not as sensitive as the AlphaScreen® measurements). Similar to the AlphaScreen® results (Fig. 1), the urea gels show that Aβ 1–40 dominates the earliest stage of the reaction and that Aβ 1–42 production starts after Aβ 1–40 (Fig. 2 A). The longer more hydrophobic Aβ products are below detection limits in the earliest stage of reaction, and then gradually accumulate with the reaction progress in time. Ultimately, at the late stage of the reaction the longer Aβ products become comparable to Aβ 1–40 and Aβ 1–42 (Fig. 2B). Thus, the longer more hydrophobic Aβ products observed in the late stage of the reaction are not transient catalytic intermediates, but products of an incomplete sequence of the processive cleavages (Fig. S1). This shift to the longer Aβ products can explain the observed drop in Aβ 1–40 and Aβ 1–42 production at the late stage of reaction (Fig. 1). In summary, we conclude that the reaction progress in time can affect the enzyme's ability to process the longer more hydrophobic Aβ peptides to Aβ 1–40 and Aβ 1–42 (different examples of factors that control processing and accumulation of reaction intermediates are illustrated in more details in Fig. S1 and on p.145 in ref. [62]).


Modulation of γ-secretase activity by multiple enzyme-substrate interactions: implications in pathogenesis of Alzheimer's disease.

Svedružić ZM, Popović K, Smoljan I, Sendula-Jengić V - PLoS ONE (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316526&req=5

pone-0032293-g002: 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.
Mentions: We further analyzed the early stage of the reaction using urea gels (Fig. 2 A–B) that can separate Aβ 1–40 and Aβ 1–42 from the other Aβ 1-x products (the urea gels are not as sensitive as the AlphaScreen® measurements). Similar to the AlphaScreen® results (Fig. 1), the urea gels show that Aβ 1–40 dominates the earliest stage of the reaction and that Aβ 1–42 production starts after Aβ 1–40 (Fig. 2 A). The longer more hydrophobic Aβ products are below detection limits in the earliest stage of reaction, and then gradually accumulate with the reaction progress in time. Ultimately, at the late stage of the reaction the longer Aβ products become comparable to Aβ 1–40 and Aβ 1–42 (Fig. 2B). Thus, the longer more hydrophobic Aβ products observed in the late stage of the reaction are not transient catalytic intermediates, but products of an incomplete sequence of the processive cleavages (Fig. S1). This shift to the longer Aβ products can explain the observed drop in Aβ 1–40 and Aβ 1–42 production at the late stage of reaction (Fig. 1). In summary, we conclude that the reaction progress in time can affect the enzyme's ability to process the longer more hydrophobic Aβ peptides to Aβ 1–40 and Aβ 1–42 (different examples of factors that control processing and accumulation of reaction intermediates are illustrated in more details in Fig. S1 and on p.145 in ref. [62]).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Biochemistry, Faculty of Medicine, University of Rijeka, Rab, Croatia. zsvedruz@biol.pmf.hr

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.

Show MeSH
Related in: MedlinePlus