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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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Related in: MedlinePlus

Steps in the catalytic cycle of γ-secretase. The model illustrates the basic biophysical principles of processive cleavages and intramembrane proteolysis [11], [37], [40], [48], [49], [51]. C99 substrate can be shown as a transmembrane helix [42], while γ-secretase can be shown as a bowl-shaped membrane-imbedded complex with its active site aspartates in the central aqueous cavity [11], [18], [19]. The initial AICD cleavage (Fig. 1) takes place between amino acids 48–49 or 49–50 [37], just under the membrane surface [42], 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 [51]–[55], [57]. 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 [51]–[54], [57]. There is no need for active use of cell's energy. The result is a sequence of processive cleavages of hydrophobic tri-peptides [48] that does not require a full exposure of the hydrophobic substrate to the aqueous catalytic site [11]. 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 [37], [40], [48], [49]. 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) [62], while the fully extended structures (C7b) can not be stabile due to unsatisfied hydrogen bonds in the peptide backbone [62]. 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 [93], apo-lipoprotein E [5], PrP C [94], or some other surface proteins [60].
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pone-0032293-g010: Steps in the catalytic cycle of γ-secretase. The model illustrates the basic biophysical principles of processive cleavages and intramembrane proteolysis [11], [37], [40], [48], [49], [51]. C99 substrate can be shown as a transmembrane helix [42], while γ-secretase can be shown as a bowl-shaped membrane-imbedded complex with its active site aspartates in the central aqueous cavity [11], [18], [19]. The initial AICD cleavage (Fig. 1) takes place between amino acids 48–49 or 49–50 [37], just under the membrane surface [42], 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 [51]–[55], [57]. 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 [51]–[54], [57]. There is no need for active use of cell's energy. The result is a sequence of processive cleavages of hydrophobic tri-peptides [48] that does not require a full exposure of the hydrophobic substrate to the aqueous catalytic site [11]. 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 [37], [40], [48], [49]. 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) [62], while the fully extended structures (C7b) can not be stabile due to unsatisfied hydrogen bonds in the peptide backbone [62]. 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 [93], apo-lipoprotein E [5], PrP C [94], or some other surface proteins [60].

Mentions: There is a standing debate whether pathological increase in Aβ42/Aβ40 ratio is a result of “a gain of function for production of Aβ42”, or “a loss of function for production of Aβ40” [20]. We find that increase in Aβ42/Aβ40 ratio can be caused by: i) increase in Aβ 1–42 production due to progress of γ-secretase reaction from pre-steady-state to steady-state catalysis (Fig. 1 and 2), ii) decrease in Aβ 1–40 production due to enzyme saturation with its C99 substrates (Fig. 3–4 and Fig. S3). In both cases, increase in Aβ42/Aβ40 ratio and decrease in Aβ40 production correlates with increase in production of the longer more hydrophobic Aβ products. The presented results are consistent with the earlier studies [37], [38], [40], [48]–[50]. The molecular mechanisms that can lead to such changes are elaborated in detail in Fig. 10 and Fig. 11. The increase in Aβ42 production can be attributed to changes in γ-secretase-C99 interaction, so that the initial cleavage takes place between the amino acids 48–49 rather than between 49–50 (Fig. 10). The increase in the longer more hydrophobic Aβ products can be attributed to decreased ability of γ-secretase to hold and fully process the nascent Aβ catalytic intermediates (Fig. 10 and Fig. 11).


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)

Steps in the catalytic cycle of γ-secretase. The model illustrates the basic biophysical principles of processive cleavages and intramembrane proteolysis [11], [37], [40], [48], [49], [51]. C99 substrate can be shown as a transmembrane helix [42], while γ-secretase can be shown as a bowl-shaped membrane-imbedded complex with its active site aspartates in the central aqueous cavity [11], [18], [19]. The initial AICD cleavage (Fig. 1) takes place between amino acids 48–49 or 49–50 [37], just under the membrane surface [42], 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 [51]–[55], [57]. 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 [51]–[54], [57]. There is no need for active use of cell's energy. The result is a sequence of processive cleavages of hydrophobic tri-peptides [48] that does not require a full exposure of the hydrophobic substrate to the aqueous catalytic site [11]. 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 [37], [40], [48], [49]. 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) [62], while the fully extended structures (C7b) can not be stabile due to unsatisfied hydrogen bonds in the peptide backbone [62]. 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 [93], apo-lipoprotein E [5], PrP C [94], or some other surface proteins [60].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0032293-g010: Steps in the catalytic cycle of γ-secretase. The model illustrates the basic biophysical principles of processive cleavages and intramembrane proteolysis [11], [37], [40], [48], [49], [51]. C99 substrate can be shown as a transmembrane helix [42], while γ-secretase can be shown as a bowl-shaped membrane-imbedded complex with its active site aspartates in the central aqueous cavity [11], [18], [19]. The initial AICD cleavage (Fig. 1) takes place between amino acids 48–49 or 49–50 [37], just under the membrane surface [42], 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 [51]–[55], [57]. 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 [51]–[54], [57]. There is no need for active use of cell's energy. The result is a sequence of processive cleavages of hydrophobic tri-peptides [48] that does not require a full exposure of the hydrophobic substrate to the aqueous catalytic site [11]. 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 [37], [40], [48], [49]. 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) [62], while the fully extended structures (C7b) can not be stabile due to unsatisfied hydrogen bonds in the peptide backbone [62]. 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 [93], apo-lipoprotein E [5], PrP C [94], or some other surface proteins [60].
Mentions: There is a standing debate whether pathological increase in Aβ42/Aβ40 ratio is a result of “a gain of function for production of Aβ42”, or “a loss of function for production of Aβ40” [20]. We find that increase in Aβ42/Aβ40 ratio can be caused by: i) increase in Aβ 1–42 production due to progress of γ-secretase reaction from pre-steady-state to steady-state catalysis (Fig. 1 and 2), ii) decrease in Aβ 1–40 production due to enzyme saturation with its C99 substrates (Fig. 3–4 and Fig. S3). In both cases, increase in Aβ42/Aβ40 ratio and decrease in Aβ40 production correlates with increase in production of the longer more hydrophobic Aβ products. The presented results are consistent with the earlier studies [37], [38], [40], [48]–[50]. The molecular mechanisms that can lead to such changes are elaborated in detail in Fig. 10 and Fig. 11. The increase in Aβ42 production can be attributed to changes in γ-secretase-C99 interaction, so that the initial cleavage takes place between the amino acids 48–49 rather than between 49–50 (Fig. 10). The increase in the longer more hydrophobic Aβ products can be attributed to decreased ability of γ-secretase to hold and fully process the nascent Aβ catalytic intermediates (Fig. 10 and Fig. 11).

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