TY - JOUR
T1 - Alzheimer amyloid precursor aspartyl proteinase activity in CHAPSO homogenates of Spodoptera frugiperda cells
AU - Carter, T.L.
AU - Verdile, G.
AU - Groth, D.
AU - Bogush, A.
AU - Thomas, S.
AU - Shen, P.
AU - Fraser, P.E.
AU - Mathews, P.
AU - Nixon, R.A.
AU - Ehrlich, M.E.
AU - Kwok, J.B.J.
AU - St. George-Hyslop, P.
AU - Schofield, P.
AU - Li, Y.
AU - Yang, A.
AU - Martins, Ralph
AU - Gandy, S.
PY - 2004
Y1 - 2004
N2 - AB Presenilins are polytopic, integral proteins that control intramembranous proteolysis at the "[gamma]-" and "[varepsilon]-" cleavage sites of the Alzheimer amyloid-[beta] precursor protein (APP) to yield amyloid-[beta] peptide (A[beta]) and the APP intracellular domain (AICD). We have overexpressed a constitutively active, pathogenic form of PS1 (known as PS1 [DELTA] exon 9) together with its substrate, APP-C99, in Spodoperta frugiperda (Sf9) cells. Sf9 cells have been reported to lack endogenous [gamma]-secretase, an unexpected finding since there exists an insect homologue of PS1. In our hands, neither intact insect cells coexpressing PS1 [DELTA] exon 9/APP-C99 nor the aqueous homogenates of these cells displayed obvious products of the [gamma]- or [varepsilon]-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 [gamma]-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.
AB - AB Presenilins are polytopic, integral proteins that control intramembranous proteolysis at the "[gamma]-" and "[varepsilon]-" cleavage sites of the Alzheimer amyloid-[beta] precursor protein (APP) to yield amyloid-[beta] peptide (A[beta]) and the APP intracellular domain (AICD). We have overexpressed a constitutively active, pathogenic form of PS1 (known as PS1 [DELTA] exon 9) together with its substrate, APP-C99, in Spodoperta frugiperda (Sf9) cells. Sf9 cells have been reported to lack endogenous [gamma]-secretase, an unexpected finding since there exists an insect homologue of PS1. In our hands, neither intact insect cells coexpressing PS1 [DELTA] exon 9/APP-C99 nor the aqueous homogenates of these cells displayed obvious products of the [gamma]- or [varepsilon]-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 [gamma]-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.
M3 - Article
SN - 0893-0341
VL - 18
SP - 261
EP - 263
JO - Alzheimer Disease and Associated Disorders
JF - Alzheimer Disease and Associated Disorders
IS - 4
ER -