Alzheimer amyloid precursor aspartyl proteinase activity in CHAPSO homogenates of Spodoptera frugiperda cells Journal Article
| Authors: | 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. |
| Article Title: | Alzheimer amyloid precursor aspartyl proteinase activity in CHAPSO homogenates of Spodoptera frugiperda cells |
| 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. |
| Keywords: | unclassified drug; nonhuman; protein domain; mass spectrometry; animal cell; animals; gene overexpression; protein degradation; cell line; membrane proteins; enzyme activity; recombinant proteins; immunoprecipitation; spodoptera; protein structure, tertiary; protein isoforms; alzheimer disease; amyloid precursor protein; gamma secretase; presenilin 1; presenilin-1; aspartic proteinase; enzyme; amyloid beta protein; detergents; insect cell; amyloid beta-protein precursor; lepidoptera; aspartic endopeptidases; humans; priority journal; article; 3 [(3 cholamidopropyl)dimethylammonio] 2 hydroxy 1 propanesulfonic acid; aspartic proteinase inhibitor; epsilon secretase; cholic acids |
| Journal Title: | Alzheimer Disease & Associated Disorders |
| Volume: | 18 |
| Issue: | 4 |
| ISSN: | 0893-0341 |
| Publisher: | Lippincott Williams & Wilkins |
| Date Published: | 2004-10-01 |
| Start Page: | 261 |
| End Page: | 263 |
| Language: | English |
| PROVIDER: | scopus |
| PUBMED: | 15592142 |
| DOI/URL: | |
| Notes: | Alzheimer Dis. Assoc. Disord. -- Cited By (since 1996):5 -- Export Date: 16 June 2014 -- CODEN: ADADE C2 - 15592142 -- Source: Scopus |
