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The amyloid-beta forming tripeptide cleavage mechanism of γ -secretase

View Article: PubMed Central - PubMed

ABSTRACT

γ-secretase is responsible for the proteolysis of amyloid precursor protein (APP) into short, aggregation-prone amyloid-beta (Aβ) peptides, which are centrally implicated in the pathogenesis of Alzheimer’s disease (AD). Despite considerable interest in developing γ-secretase targeting therapeutics for the treatment of AD, the precise mechanism by which γ-secretase produces Aβ has remained elusive. Herein, we demonstrate that γ-secretase catalysis is driven by the stabilization of an enzyme-substrate scission complex via three distinct amino-acid-binding pockets in the enzyme’s active site, providing the mechanism by which γ-secretase preferentially cleaves APP in three amino acid increments. Substrate occupancy of these three pockets occurs after initial substrate binding but precedes catalysis, suggesting a conformational change in substrate may be required for cleavage. We uncover and exploit substrate cleavage preferences dictated by these three pockets to investigate the mechanism by which familial Alzheimer’s disease mutations within APP increase the production of pathogenic Aβ species.

Doi:: http://dx.doi.org/10.7554/eLife.17578.001

No MeSH data available.


Related in: MedlinePlus

HEK cell expression and Aβ production from consecutive phenylalanine APP mutants.(A) Aβ38, Aβ40 and Aβ42 levels secreted from HEK cells after transient transfection with APP mutants containing stretches of consecutive phenylalanines (see Figure 6D). Aβ levels measured by 4G8 ELISA. Mean ± SD, n = 3. (B) Expression levels of consecutive phenylalanine APP mutants from Figure 6D.DOI:http://dx.doi.org/10.7554/eLife.17578.010
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fig6s1: HEK cell expression and Aβ production from consecutive phenylalanine APP mutants.(A) Aβ38, Aβ40 and Aβ42 levels secreted from HEK cells after transient transfection with APP mutants containing stretches of consecutive phenylalanines (see Figure 6D). Aβ levels measured by 4G8 ELISA. Mean ± SD, n = 3. (B) Expression levels of consecutive phenylalanine APP mutants from Figure 6D.DOI:http://dx.doi.org/10.7554/eLife.17578.010

Mentions: Next, we attempted to determine how many phenylalanines in a row γ-secretase was capable of skipping by taking advantage of the fact that the GG motif apparently allows for γ-secretase to deviate from normal sequential tripeptide cleavage. Astonishingly, even after increasing the number of phenylalanines in a row to eight, γ-secretase was still able to produce Aβ above mock transfected levels (Figure 6D,E). The V44F⇒M51F mutant produced mostly Aβ38 (Figure 6—figure supplement 1), in what may be a single endoproteolytic cleavage event, although we have not been able to obtain enough AICD for MS confirmation. Predictably, the Aβ42/40 ratios for these mutants follow a pattern expected if γ-secretase cleaves at the next available site after each additional Phe (Figure 6F).


The amyloid-beta forming tripeptide cleavage mechanism of γ -secretase
HEK cell expression and Aβ production from consecutive phenylalanine APP mutants.(A) Aβ38, Aβ40 and Aβ42 levels secreted from HEK cells after transient transfection with APP mutants containing stretches of consecutive phenylalanines (see Figure 6D). Aβ levels measured by 4G8 ELISA. Mean ± SD, n = 3. (B) Expression levels of consecutive phenylalanine APP mutants from Figure 6D.DOI:http://dx.doi.org/10.7554/eLife.17578.010
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5134833&req=5

fig6s1: HEK cell expression and Aβ production from consecutive phenylalanine APP mutants.(A) Aβ38, Aβ40 and Aβ42 levels secreted from HEK cells after transient transfection with APP mutants containing stretches of consecutive phenylalanines (see Figure 6D). Aβ levels measured by 4G8 ELISA. Mean ± SD, n = 3. (B) Expression levels of consecutive phenylalanine APP mutants from Figure 6D.DOI:http://dx.doi.org/10.7554/eLife.17578.010
Mentions: Next, we attempted to determine how many phenylalanines in a row γ-secretase was capable of skipping by taking advantage of the fact that the GG motif apparently allows for γ-secretase to deviate from normal sequential tripeptide cleavage. Astonishingly, even after increasing the number of phenylalanines in a row to eight, γ-secretase was still able to produce Aβ above mock transfected levels (Figure 6D,E). The V44F⇒M51F mutant produced mostly Aβ38 (Figure 6—figure supplement 1), in what may be a single endoproteolytic cleavage event, although we have not been able to obtain enough AICD for MS confirmation. Predictably, the Aβ42/40 ratios for these mutants follow a pattern expected if γ-secretase cleaves at the next available site after each additional Phe (Figure 6F).

View Article: PubMed Central - PubMed

ABSTRACT

γ-secretase is responsible for the proteolysis of amyloid precursor protein (APP) into short, aggregation-prone amyloid-beta (Aβ) peptides, which are centrally implicated in the pathogenesis of Alzheimer’s disease (AD). Despite considerable interest in developing γ-secretase targeting therapeutics for the treatment of AD, the precise mechanism by which γ-secretase produces Aβ has remained elusive. Herein, we demonstrate that γ-secretase catalysis is driven by the stabilization of an enzyme-substrate scission complex via three distinct amino-acid-binding pockets in the enzyme’s active site, providing the mechanism by which γ-secretase preferentially cleaves APP in three amino acid increments. Substrate occupancy of these three pockets occurs after initial substrate binding but precedes catalysis, suggesting a conformational change in substrate may be required for cleavage. We uncover and exploit substrate cleavage preferences dictated by these three pockets to investigate the mechanism by which familial Alzheimer’s disease mutations within APP increase the production of pathogenic Aβ species.

Doi:: http://dx.doi.org/10.7554/eLife.17578.001

No MeSH data available.


Related in: MedlinePlus