Baculoviruses expressing the human familial Alzheimer's disease presenilin 1 mutation lacking exon 9 increase levels of an amyloid beta-like protein in Sf9 cells

G. Verdile, D. Groth, P. M. Mathews, P. St George-Hyslop, P. E. Fraser, T. V. Ramabhadran, J. B J Kwok, P. R. Schofield, T. Carter, S. Gandy, R. N. Martins*

*Corresponding author for this work

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-β protein (Aβ) that is central to the pathogenesis of Alzheimer's disease. PS1 regulates the intramembranous proteolysis of a 99-amino-acid C-terminal fragment of the amyloid precursor protein (APP-C99), a cleavage event that releases Aβ following a reaction catalyzed by an enzyme termed 'γ-secretase'. The molecular mechanism of PS1-mediated, γ-secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the γ-secretase profease or whether instead PS1 mediates γ-secretase activity indirectly, perhaps by regulating the trafficking or presentation of substrates to the 'authentic' protease, which may be a molecule distinct from PS1. To address this issue, the baculovirus expression system was used to co-express: (i) APP-C99; (ii) a pathogenic, constitutively active mutant form of PS1 lacking exon 9 (PS1ΔE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an Aβ-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1ΔE9 mutation. The addition to APP-C99-infected cells of baculoviruses bearing nicastrin, also a transmembrane protein, had a neutral or inhibitory effect on the reaction; tropomyosin viruses had the same effect as nicastrin viruses. These results suggest that PS1ΔE9 molecules expressed in Sf9 cells retain the ability to modulate Aβ levels. Baculoviral-expressed PS1ΔE9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting γ-secretase activity from its individual purified component parts.

Original languageEnglish
Pages (from-to)594-602
Number of pages9
JournalMolecular Psychiatry
Volume9
Issue number6
DOIs
Publication statusPublished - Jun 2004
Externally publishedYes

Keywords

  • γ-secretase
  • Alzheimer's disease
  • Amyloid precursor protein
  • Amyloid-β
  • Nicastrin
  • Presenilin-1
  • Proteolytic processing
  • Spodoptera frugiperda cells

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