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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

AICD fragments for the three I45 FAD mutants determined by western blot using the AICD 50–99 specific antibody.(A) Western blot of the AICD 50–99 fragment for I45F, I45V and I45T FAD mutations. (B) Quantification of western blot bands from (A). Mean ± SD, n = 3, t-test **<0.01.DOI:http://dx.doi.org/10.7554/eLife.17578.013
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fig7s2: AICD fragments for the three I45 FAD mutants determined by western blot using the AICD 50–99 specific antibody.(A) Western blot of the AICD 50–99 fragment for I45F, I45V and I45T FAD mutations. (B) Quantification of western blot bands from (A). Mean ± SD, n = 3, t-test **<0.01.DOI:http://dx.doi.org/10.7554/eLife.17578.013

Mentions: V50F partially rescues the Aβ42/40 ratio when paired with I45T, but remains significantly elevated compared to WT, indicating the I45T mutant both influences initial ε cleavage and uncouples ε from γ cleavages. The change in ε cleavage preference of I45T was verified using the AICD 50–99 specific antibody (Figure 7—figure supplement 2), showing a small reduction in AICD 50–99. Interestingly, the I45T mutant reduces the amount of AICD 50–99 comparable to I45V, even though these two mutants display very different Aβ42/40 ratios. This again suggests I45T dissociates cleavage downstream of ε to achieve such a high Aβ42/40 ratio. Exactly how I45T does this is currently unknown and requires further investigation.


The amyloid-beta forming tripeptide cleavage mechanism of γ -secretase
AICD fragments for the three I45 FAD mutants determined by western blot using the AICD 50–99 specific antibody.(A) Western blot of the AICD 50–99 fragment for I45F, I45V and I45T FAD mutations. (B) Quantification of western blot bands from (A). Mean ± SD, n = 3, t-test **<0.01.DOI:http://dx.doi.org/10.7554/eLife.17578.013
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5134833&req=5

fig7s2: AICD fragments for the three I45 FAD mutants determined by western blot using the AICD 50–99 specific antibody.(A) Western blot of the AICD 50–99 fragment for I45F, I45V and I45T FAD mutations. (B) Quantification of western blot bands from (A). Mean ± SD, n = 3, t-test **<0.01.DOI:http://dx.doi.org/10.7554/eLife.17578.013
Mentions: V50F partially rescues the Aβ42/40 ratio when paired with I45T, but remains significantly elevated compared to WT, indicating the I45T mutant both influences initial ε cleavage and uncouples ε from γ cleavages. The change in ε cleavage preference of I45T was verified using the AICD 50–99 specific antibody (Figure 7—figure supplement 2), showing a small reduction in AICD 50–99. Interestingly, the I45T mutant reduces the amount of AICD 50–99 comparable to I45V, even though these two mutants display very different Aβ42/40 ratios. This again suggests I45T dissociates cleavage downstream of ε to achieve such a high Aβ42/40 ratio. Exactly how I45T does this is currently unknown and requires further investigation.

View Article: PubMed Central - PubMed

ABSTRACT

&gamma;-secretase is responsible for the proteolysis of amyloid precursor protein (APP) into short, aggregation-prone amyloid-beta (A&beta;) peptides, which are centrally implicated in the pathogenesis of Alzheimer&rsquo;s disease (AD). Despite considerable interest in developing &gamma;-secretase targeting therapeutics for the treatment of AD, the precise mechanism by which &gamma;-secretase produces A&beta; has remained elusive. Herein, we demonstrate that &gamma;-secretase catalysis is driven by the stabilization of an enzyme-substrate scission complex via three distinct amino-acid-binding pockets in the enzyme&rsquo;s active site, providing the mechanism by which &gamma;-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&rsquo;s disease mutations within APP increase the production of pathogenic A&beta; species.

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

No MeSH data available.


Related in: MedlinePlus