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 |
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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 57â59 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 â¼6-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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M005968200 |