Abstract
Gamma-secretase is an enzyme complex that mediates both Notch signaling and beta-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid beta peptide (Abeta), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of gamma-secretase gives rise to Abeta peptides of different lengths, where Abeta42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320-374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of gamma-secretase, for gamma-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, gamma-secretase complex formation, and had a differential effect on Abeta-peptide production. Although the production of Abeta38, Abeta39, and Abeta40 was severely impaired, the effect on Abeta42 was affected to a lesser extent, implying that the production of the AD-related Abeta42 peptide is separate from the production of the Abeta38, Abeta39, and Abeta40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the epsilon/S3 and gamma sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different Abeta peptides without affecting Notch processing, two parameters of significance when considering gamma-secretase as a target for pharmaceutical intervention in AD.
Highlights
Alzheimer disease (AD)2 is the most common form of dementia in elderly and is a multifactorial disease caused by a progressive
In this study we have examined the role of the large hydrophilic loop of presenilin 1 (PS1), the catalytic subunit of ␥-secretase, for ␥-secretase complex formation and activity on Notch and amyloid precursor protein (APP) processing
PS1 and PS2 share a high degree of homology and can form ␥-secretase complexes that are both active on APP and Notch processing, there are some domains that differ extensively between these two proteins, especially the large cytoplasmic loop between TMD6 and -7 [26]
Summary
CDNA Constructs—Full-length PS1wt were cloned into the pcDNA5FRT/TO vector (Invitrogen) on BamH1/Not sites. BD8:APP was transfected with 800 ng of PS1wt/ PS1⌬exon 10 or 800 ng of each NTFwt and truncated CTF, empty pcDNA5 vector was added to adjust for differences in DNA amounts, and 200 ng of CMV--gal and 100 ng of green fluorescent protein was added. Cycloheximide Treatment of Cells—BD8 and BD8:NTF cells were transfected in 1 well of a 6-well tissue culture plate with 500 ng of CMV--gal and 200 ng of green fluorescent protein as well as 2000 ng of PS1wt or PS1⌬exon 10 for BD8 cells or 2000 ng of each of CTFwt or CTF start 375 for BD8:NTF. B, expression analysis is shown of lysed BD8 or BD8:NTF cells transfected with PS1wt, PS1⌬exon 10, CTFwt, CTF start 375, and empty vector using a PS1-NTF antibody (NT-1).
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