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

Abstract

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-b protein (Ab) 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 Ab following a reaction catalyzed by an enzyme termed 'c-secretase'. The molecular mechanism of PS1-mediated, c-secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the c-secretase protease or whether instead PS1 mediates c-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 (PS1DE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an Ab-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1DE9 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 PS1DE9 molecules expressed in Sf9 cells retain the ability to modulate Ab levels. Baculoviral-expressed PS1DE9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting c-secretase activity from its individual purified component parts.

Efficient production of a mature and functional gamma secretase protease

“…The activity of the recombinant PS1(wt)-γ-secretase and PS1(Δ9) was evaluated in cell free assays. PS1(Δ9) does not require other core γ -secretase components to process γ-secretase substrates 26 , 44 , 45 . Cell free assays were prepared with both APP-C99 or Notch-C100 substrates and the Aβ/AICD (APP intracellular domain) and NICD (Notch intracellular domain) proteolytic cleavage fragments were detected by immunoblotting 54 , 55 .…”

“…The PS1 mutation-PS1(Δ9) is of particular interest in understanding the molecular mechanisms of γ-secretase mediated proteolysis. PS1(Δ9) does not need endoproteolytic activation (lacks exon 9 wherein lies the endoproteolytic site), to process γ-secretase substrates 26 , 44 , 45 .…”

The structure and function of Alzheimer’s gamma secretase enzyme complex

“…However, there are many factors including diet, hormonal status, and genetic mutations that influence the processing of APP to generate Aβ (reviewed in Refs. 3,4 ). Two additional cleavage sites on the APP-C terminal fragment have been identified and termed the ε-cleavage site and ζ-cleavage site.…”

Design of signal peptide bombyxin and its effect on secretory expression efficiency and levels of Helicobacter pylori urease subunit B in silkworm cells and larvae

“…The absorbance values of the rUreB band without the signal peptide were 120 ± 16, whereas the value for the rUreB band fused to the signal peptide was 183 ± 27; thus, the expression level in silkworm larvae was approximately 1.5-fold higher when the bombyxin signal peptide was included. Various types of genetic manipulation techniques and alterative technologies have been implemented to improve expression levels for the mass production of proteins in insect cells, including site-directed and random mutagenesis (Verdile et al 2004), promoter exchange and signal peptide insertion (Golden et al 1998), and increased plasmid copy number (Deml et al 1999). The secretory expression of recombinant proteins in host cells is also a highly effective strategy.…”

“…was designed using SignalP 3.0 (Verdile et al 2004). The probability of this sequence being a signal peptide is 1.000, and the maximum cleavage site probability is 0.995.…”