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P4‐071: The Amyloid‐B Generating Tri‐Peptide Cleavage Mechanism of Gamma‐Secretase: Implications for Alzheimer's Disease

2016; Wiley; Volume: 12; Issue: 7S_Part_21 Linguagem: Inglês

10.1016/j.jalz.2016.06.2161

1552-5279

Michael S. Wolfe, David M. Bolduc, Daniel R. Montagna, Matthew Seghers, Dennis J. Selkoe,

Peptidase Inhibition and Analysis

γ-Secretase produces not only Aβ40 and Aβ42 but other Aβ peptides ranging from 38 to 49 residues. The protease first cleaves APP substrate within its transmembrane domain (TMD) at ε sites to form intracellular domain (AICD) and either Aβ48 or Aβ49. Subsequent cleavage from the carboxy terminus occurs along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42. We sought to elucidate the unknown mechanism that dictates the sites of endoproteolytic and carboxypeptidase cleavage of APP by γ-secretase. Human γ-secretase was isolated and solubilized from mammalian cells over-expressing all four protease components. After incubation with APP substrate C100Flag and mutations thereof, AICD-Flag was analyzed by western blot and Aβ40 and Aβ42 were quantified by specific ELISAs. In other experiments, HEK293 cells were transiently transfected with APP and mutations thereof, followed by analysis of the media via Aβ40 and Aβ42 ELISAs. Tripeptides representing carboxypeptidase trimming products from the APP transmembrane domain inhibited γ-secretase activity noncompetitively. Substrate mutation to Phe could drive γ-secretase cleavage toward either the Aβ49→Aβ40 pathway or the Aβ48→Aβ42 pathway consistent with an inability of Phe to be accommodated in the S2' pocket relative to ε cleavage. Mutation of a second Phe in the S2' pocket relative to carboxypeptidase trimming sites overrode effects of Phe mutations near the ε site. Multiple Phe mutations that dramatically reduce proteolysis result in stabilized substrate binding to the γ-secretase complex. A Phe mutation near the ε cleavage site that favors the Aβ49→Aβ40 pathway rescued the increased Aβ42/Aβ40 caused by familial AD mutations in APP. These results suggest that upon initial binding of APP substrate to the γ-secretase complex, the helical transmembrane domain unwinds to bind to three hydrophobic pockets (S1', S2' and S3'), setting up a high-energy enzyme-substrate complex poised for amide bond hydrolysis. Blocking helix unwinding and binding to the S1'-S2'-S3' pockets stabilizes an initial enzyme-substrate complex. The two-step process of initial binding followed by unwinding and binding to S1'-S2'-S3' pockets is also consistent with the noncompetitive inhibition mechanism of tripeptide byproducts. Familial AD mutations in APP apparently increase Aβ42/Aβ40 primarily by affecting the site of ε cleavage.