Substrate interaction inhibits γ-secretase production of amyloid-β peptides.
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ABSTRACT:
SUBMITTER: Zhao J
PROVIDER: S-EPMC8219260 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Production of amyloid β-protein (Aβ) is carried out by the membrane-embedded γ-secretase complex. Mutations in the transmembrane domain of amyloid β-protein precursor (APP) associated with early-onset familial Alzheimer's disease (FAD) can alter the ratio of aggregation-prone 42-residue Aβ (Aβ42) to 40-residue Aβ (Aβ40). However, APP substrate is proteolyzed processively by γ-secretase along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38. Effects of FAD mutations on each proteolytic step are unknown, largely due to difficulties in detecting and quantifying longer Aβ peptides. To address this, we carried out systematic and quantitative analyses of all tri- and tetrapeptide coproducts from proteolysis of wild-type and 14 FAD-mutant APP substrates by purified γ-secretase. These small peptides, including FAD-mutant forms, were detected by tandem mass spectrometry and quantified by establishing concentration curves for each of 32 standards. APP intracellular domain (AICD) coproducts were quantified by immunoblot, and the ratio of AICD products corresponding to Aβ48 and Aβ49 was determined by mass spectrometry. Levels of individual Aβ peptides were determined by subtracting levels of peptide coproducts associated with degradation from those associated with production. This method was validated for Aβ40 and Aβ42 by specific ELISAs and production of equimolar levels of Aβ and AICD. Not all mutant substrates led to increased Aβ42/40. However, all 14 disease-causing mutations led to inefficient processing of longer forms of Aβ ≥ 45 residues. In addition, the effects of certain mutations provided insight into the mechanism of processive proteolysis: intermediate Aβ peptides apparently remain bound for subsequent trimming and are not released and reassociated.
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Project description:gamma-Secretase is a membrane protein complex that cleaves the beta-amyloid precursor protein (APP) within the transmembrane region, after prior processing by beta-secretase, producing amyloid beta-peptides Abeta(40) and Abeta(42). Errant production of Abeta-peptides that substantially increases Abeta(42) production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1, which contains the proteolytic active site, and three other membrane proteins [nicastrin, anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 (PEN-2)] are required to form the core of the active gamma-secretase complex. Here, we report the purification of the native gamma-secretase complexes from HeLa cell membranes and the identification of an additional gamma-secretase complex subunit, CD147, a transmembrane glycoprotein with two Ig-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through coimmunoprecipitation studies of the purified protein from HeLa cells and of solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of Abeta peptides without changing the expression level of the other gamma-secretase components or APP substrates whereas CD147 overexpression had no statistically significant effect on Abeta-peptide production, other gamma-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the gamma-secretase complex down-modulates the production of Abeta-peptides.
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