Publications

Abstract

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-beta protein (Abeta) 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 Abeta following a reaction catalyzed by an enzyme termed 'gamma-secretase'. The molecular mechanism of PS1-mediated, gamma-secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the gamma-secretase protease or whether instead PS1 mediates gamma-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 (PS1DeltaE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an Abeta-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1DeltaE9 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 PS1DeltaE9 molecules expressed in Sf9 cells retain the ability to modulate Abeta levels. Baculoviral-expressed PS1DeltaE9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting gamma-secretase activity from its individual purified component parts.