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.
Verdile
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Sir James McCusker Alzheimer's Disease Research Unit, University of Western Australia, School of Psychiatry and Clinical Neurosciences, Hollywood Private Hospital, Nedlands, WA, Australia.
Groth
D
D
Mathews
P M
PM
St George-Hyslop
P
P
Fraser
P E
PE
Ramabhadran
T V
TV
Kwok
J B J
JB
Schofield
P R
PR
Carter
T
T
Gandy
S
S
Martins
R N
RN
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