Abstract
Intramembranous "gamma-secretase" processing of beta-amyloid precursor protein (APP) and other transmembrane proteins, including Notch, is mediated by a macromolecular complex consisting of presenilins (PSs), nicastrin (NCT), APH-1, and PEN-2. We now demonstrate that in cells coexpressing PS1, APH-1, and NCT, full-length PS1 accumulates to high levels and is fairly stable. Upon expression of PEN-2, the levels of PS1 holoprotein are significantly reduced, commensurate with an elevation in levels of PS1 fragments. These findings suggest that APH-1 and NCT are necessary for stabilization of full-length PS1 and that PEN-2 is critical for the proteolysis of stabilized PS1. In N2a and 293 cell lines that stably overexpress PS1, APH-1, NCT, and PEN-2, PS1 fragment levels are elevated by up to 10-fold over endogenous levels. In these cells, we find a marked accumulation of the APP-CTF gamma (AICD) fragment and a concomitant reduction in levels of both APP-CTF beta and CTF alpha. Moreover, the production of the gamma-secretase-generated Notch S3/NICD derivative is modestly elevated. However, we failed to observe a corresponding increase in levels of secreted A beta peptides in the medium of these cells. These results lead us to conclude that, although the PS1, APH-1, NCT, and PEN-2 are essential for gamma-secretase activity, the proteolysis of APP-CTF and Notch S2/NEXT are differentially regulated and require the activity of additional cofactors that promote production of AICD, NICD, and A beta.
Highlights
Similar results were obtained for PEN-2; the steady-state levels of PEN-2 are higher when coexpressed with APH-1␣ compared with settings in which only PEN-2 is expressed (Fig. 1A, panel II, compare lanes 5 and 7; 8 and 9)
Our results offer the suggestion that APH-1 serves as a scaffold protein that stabilizes full-length PS1 and NCT and that PEN-2 is an essential component of the apparatus that is responsible for PS1 endoproteolysis
It is well established that the macromolecular complex that includes PS1 is responsible for intramembranous processing of amyloid precursor protein loid precursor protein (APP)-cells. These terminal fragments (CTF) and Notch S2/NEXT to generate A peptides and S3/NICD, respectively
Summary
In N2a and 293 cell lines that stably overexpress PS1, APH-1, NCT, and PEN-2, PS1 fragment levels are elevated by up to 10-fold over endogenous levels. When PEN-2 was expressed in addition to PS1, APH-1␣, and NCT, the levels of PS1 fragments were elevated even further, commensurate with a marked reduction in levels of full-length PS1 (Fig. 1A, panel IV, lane 9).
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