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.
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.
Sir James McCusker Alzheimer's Disease Research Unit, University of Western Australia, School of Psychiatry and Clinical Neurosciences, Hollywood Private Hospital, Nedlands, WA, Australia.
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Verdile
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Groth
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Mathews
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St George-Hyslop
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Fraser
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Ramabhadran
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JB
Kwok
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Schofield
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Carter
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Gandy
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Martins
D000544:Alzheimer Disease / Q000235:genetics*; D016229:Amyloid beta-Peptides / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D005091:Exons / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D053764:Presenilin-1; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera; D014162:Transfection
D016229:Amyloid beta-Peptides; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1; D011994:Recombinant Proteins