Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid  peptide (A). The A protein precursor (APP) is cleaved by -secretase to generate a C-terminal fragment, CTF, which in turn is cleaved by ␥-secretase to generate A. Alternative cleavage of the APP by ␣-secretase at A16/17 generates the C-terminal fragment, CTF␣. In addition to A, endoproteolytic cleavage of CTF␣ and CTF by ␥-secretase should yield a C-terminal fragment of 57-59 residues (CTF␥). However, CTF␥ 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 A as well as an ϳ6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTF␥, and we describe an in vitro assay for ␥-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF␥ fragment. Three compounds previously identified as ␥-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 CTF␥, providing additional evidence that the fragment arises from ␥-secretase cleavage. Consistent with reports that presenilins are the elusive ␥-secretases, subcellular fractionation studies showed that presenilin-1, CTF␣, and CTF are enriched in the CTF␥-generating fractions. The in vitro ␥-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for ␥-secretase inhibitors.
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