A Novel γ-Secretase Assay Based on Detection of the Putative C-terminal Fragment-γ of Amyloid β Protein Precursor

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid β peptide (Aβ). The Aβ protein precursor (APP) is cleaved by β-secretase to generate a C-terminal fragment, CTFβ, which in turn is cleaved by γ-secretase to generate Aβ. Alternative cleavage of the APP by α-secret...

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Veröffentlicht in:The Journal of biological chemistry 2001-01, Vol.276 (1), p.481
Hauptverfasser: Inga Pinnix, Usha Musunuru, Han Tun, Arati Sridharan, Todd Golde, Christopher Eckman, Chewki Ziani-Cherif, Luisa Onstead, Kumar Sambamurti
Format: Artikel
Sprache:eng
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Zusammenfassung:Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid β peptide (Aβ). The Aβ protein precursor (APP) is cleaved by β-secretase to generate a C-terminal fragment, CTFβ, which in turn is cleaved by γ-secretase to generate Aβ. Alternative cleavage of the APP by α-secretase at Aβ16/17 generates the C-terminal fragment, CTFα. In addition to Aβ, endoproteolytic cleavage of CTFα and CTFβ by γ-secretase should yield a C-terminal fragment of 57–59 residues (CTFγ). However, CTFγ has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo . We detected the in vitro generation of Aβ as well as an ∼6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTFγ, and we describe an in vitro assay for γ-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTFγ fragment. Three compounds previously identified as γ-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone ( t -butoxycarbonyl-Val-Ile-( S )-4-amino-3-oxo-2,2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTFγ, providing additional evidence that the fragment arises from γ-secretase cleavage. Consistent with reports that presenilins are the elusive γ-secretases, subcellular fractionation studies showed that presenilin-1, CTFα, and CTFβ are enriched in the CTFγ-generating fractions. The in vitro γ-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for γ-secretase inhibitors.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M005968200