Alzheimer amyloid precursor aspartyl proteinase activity in CHAPSO homogenates of Spodoptera frugiperda cells.
Presenilins are polytopic, integral proteins that control intramembranous proteolysis at the "gamma-" and "epsilon-" cleavage sites of the Alzheimer amyloid-beta precursor protein (APP) to yield amyloid-beta peptide (Abeta) 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 Spodoptera 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 epsilon-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.
Carter
Troy L
TL
Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Verdile
Giuseppe
G
Groth
David
D
Bogush
Alexey
A
Thomas
Stefani
S
Shen
Patrick
P
Fraser
Paul E
PE
Mathews
Paul
P
Nixon
Ralph A
RA
Ehrlich
Michelle E
ME
Kwok
John B J
JB
St George-Hyslop
Peter
P
Schofield
Peter
P
Li
Yueming
Y
Yang
Austin
A
Martins
Ralph N
RN
Gandy
Sam
S
D000544:Alzheimer Disease / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002460:Cell Line; D002793:Cholic Acids / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D053764:Presenilin-1; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D016564:Amyloid beta-Protein Precursor; D002793:Cholic Acids; D003902:Detergents; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1; D020033:Protein Isoforms; D011994:Recombinant Proteins; C048531:chapso; D016282:Aspartic Acid Endopeptidases