A novel gamma-secretase assay based on detection of the putative C-terminal fragment-gamma of amyloid beta protein precursor

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid b eta peptide (A beta). The A beta protein precursor (APP) is cleaved by beta -secretase to, generate a C-terminal fragment, CTF beta, which in turn is cleaved by gamma -secretase to generate A beta. Alternative cleavage of the APP by alpha -secretase at A beta 16/17 generates the C-terminal fragment, CTF alpha. In addition to AP, endoproteolytic cleavage of CTF alpha and CT F beta by gamma -secretase should yield a C-terminal fragment of 57-59 resi dues (CTF gamma), However, CTF gamma has not yet been reported in either br ain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of A beta as well as an similar to6-kDa fragment f rom guinea pig brain membranes. We have provided biochemical and pharmacolo gical evidence that this 6-kDa fragment is the elusive CTF gamma, and we de scribe an in vitro assay for gamma -secretase activity. The fragment migrat es with a synthetic peptide corresponding to the 57-residue CTF gamma fragm ent. Three cornpounds previously identified as gamma -secretase inhibitors, pepstatin-Al MG132, and a substrate-based dlifluoroketone (t-butoxycarbony l-Val-Ile-(S)-4-amino-3-difluoropentanoyl-Val-Ile-OMe), reduced the yield o f CTF gamma, providing additional evidence that the fragment arises from ga mma -secretase cleavage. Consistent with reports that presenilins are the e lusive gamma -secretases, subcellular fractionation studies showed that pre senilin-1, CTF alpha, and CTF beta are enriched in the CTF gamma -generatin g fractions. The in vitro gamma -secretase assay described here will be use ful for the detailed characterization of the enzyme and to screen for gamma -secretase inhibitors.