Abstract
Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.
Highlights
Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of ␥-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins
GSIs have been repurposed for various cancers, largely based on the premise that they act as inhibitors of Notch1 signaling [14, 15]
When the DKO cell was co-transfected with APP Swedish mutant (APPsw) and either PSEN1, PSEN2, or both, the total A levels were equivalent (Fig. 1F)
Summary
Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of ␥-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular -amyloid than does PS1. We found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. ␥-secretase cleavage mediates complex signaling events through cleavage of multiple transmembrane proteins on both tumor cells and normal cells, and there remain large gaps in our knowledge regarding how GSIs alter specific physiologic and pathophysiologic signaling events
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