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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

Secreted Aβ levels from V40F, A42F and V44F.(A) Aβ38, Aβ40 and Aβ42 levels measured from V40F, A42F and V44F transfected HEK cells respectively, as well as mock transfected and WT. Aβ levels measured by 6E10 ELISA. Mean ± SD, n = 3.DOI:http://dx.doi.org/10.7554/eLife.17578.014
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fig7s3: Secreted Aβ levels from V40F, A42F and V44F.(A) Aβ38, Aβ40 and Aβ42 levels measured from V40F, A42F and V44F transfected HEK cells respectively, as well as mock transfected and WT. Aβ levels measured by 6E10 ELISA. Mean ± SD, n = 3.DOI:http://dx.doi.org/10.7554/eLife.17578.014

Mentions: Although we are unable to measure every cleavage product from the ~70 mutant forms of APP we generated, we do note that every time a Phe was placed in the P2’ position of a cleavage product that we could readily and directly measure, there were almost negligible amounts of that product formed. For example, in the cell-based assay, V40F, A42F and V44F generated levels of Aβ38, Aβ40 and Aβ42, respectively, that were actually less than mock transfected levels (Figure 7—figure supplement 3). These Aβ levels are orders of magnitude less than that from WT APP transfected cells, although we cannot say whether these low levels of Aβ species were produced from endogenous HEK cell APP or from our transfected mutants. Similarly, we were unable to detect by MS any AICD products containing a Phe in the P2’ position. Together, these data suggest that aromatic amino acids may be completely excluded from the S2’ pocket and further demonstrates that substrate occupancy of the three S’ pockets is an absolute requirement for catalysis.


The amyloid-beta forming tripeptide cleavage mechanism of γ -secretase
Secreted Aβ levels from V40F, A42F and V44F.(A) Aβ38, Aβ40 and Aβ42 levels measured from V40F, A42F and V44F transfected HEK cells respectively, as well as mock transfected and WT. Aβ levels measured by 6E10 ELISA. Mean ± SD, n = 3.DOI:http://dx.doi.org/10.7554/eLife.17578.014
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Related In: Results  -  Collection

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fig7s3: Secreted Aβ levels from V40F, A42F and V44F.(A) Aβ38, Aβ40 and Aβ42 levels measured from V40F, A42F and V44F transfected HEK cells respectively, as well as mock transfected and WT. Aβ levels measured by 6E10 ELISA. Mean ± SD, n = 3.DOI:http://dx.doi.org/10.7554/eLife.17578.014
Mentions: Although we are unable to measure every cleavage product from the ~70 mutant forms of APP we generated, we do note that every time a Phe was placed in the P2’ position of a cleavage product that we could readily and directly measure, there were almost negligible amounts of that product formed. For example, in the cell-based assay, V40F, A42F and V44F generated levels of Aβ38, Aβ40 and Aβ42, respectively, that were actually less than mock transfected levels (Figure 7—figure supplement 3). These Aβ levels are orders of magnitude less than that from WT APP transfected cells, although we cannot say whether these low levels of Aβ species were produced from endogenous HEK cell APP or from our transfected mutants. Similarly, we were unable to detect by MS any AICD products containing a Phe in the P2’ position. Together, these data suggest that aromatic amino acids may be completely excluded from the S2’ pocket and further demonstrates that substrate occupancy of the three S’ pockets is an absolute requirement for catalysis.

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