Alzheimer amyloid precursor aspartyl proteinase activity in CHAPSO homogenates of Spodoptera frugiperda cells

Troy L. Carter, Giuseppe Verdile, David Groth, Alexey Bogush, Stefani Thomas, Patrick Shen, Paul E. Fraser, Paul Mathews, Ralph A. Nixon, Michelle E. Ehrlich, John B.J. Kwok, Peter St. George-Hyslop, Peter Schofield, Yueming Li, Austin Yang, Ralph N. Martins, Sam Gandy

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Abstract

Presenilins are polytopic, integral proteins that control intramembranous proteolysis at the "γ-" and "ε-" cleavage sites of the Alzheimer amyloid-β precursor protein (APP) to yield amyloid-β peptide (Aβ) and the APP intracellular domain (AICD). We have overexpressed a constitutively active, pathogenic form of PS1 (known as PS1 Δ exon 9) together with its substrate, APP-C99, in Spodoperta frugiperda (Sf9) cells. Sf9 cells have been reported to lack endogenous γ-secretase, an unexpected finding since there exists an insect homologue of PS1. In our hands, neither intact insect cells coexpressing PS1 Δ exon 9/APP-C99 nor the aqueous homogenates of these cells displayed obvious products of the γ- or ε-secretase reactions, as reported. Surprisingly, when APP-C99-expressing cells were homogenized in 3[(3-cholamidopropyl) dimethylammonio]-2- hydroxypropanesulfonic acid (CHAPSO), a detergent known to support γ-secretase activity, subsequent incubation led to the accumulation of an AICD-like peptide (AICD-L). Aspartyl proteinase inhibitors were effective in preventing the appearance of AICD-L, but inhibitors of other classes of proteinases were ineffective. Immunoprecipitation-mass spectrometry of AICD-L revealed its identity as the minor of the two known AICDs.

Original languageEnglish (US)
Pages (from-to)261-263
Number of pages3
JournalAlzheimer disease and associated disorders
Volume18
Issue number4
StatePublished - Oct 1 2004
Externally publishedYes

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Carter, T. L., Verdile, G., Groth, D., Bogush, A., Thomas, S., Shen, P., Fraser, P. E., Mathews, P., Nixon, R. A., Ehrlich, M. E., Kwok, J. B. J., St. George-Hyslop, P., Schofield, P., Li, Y., Yang, A., Martins, R. N., & Gandy, S. (2004). Alzheimer amyloid precursor aspartyl proteinase activity in CHAPSO homogenates of Spodoptera frugiperda cells. Alzheimer disease and associated disorders, 18(4), 261-263.