Regulated hyperaccumulation of presenilin-1 and the "gamma-secretase" complex. Evidence for differential intramembranous processing of transmembrane subatrates.
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.
Kim
Seong-Hun
SH
Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, Illinois 60637, USA.
Ikeuchi
Takeshi
T
Yu
Chunjiang
C
Sisodia
Sangram S
SS
D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000737:chemistry; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D053764:Presenilin-1; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D016229:Amyloid beta-Peptides; D018076:DNA, Complementary; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D010455:Peptides; D053764:Presenilin-1; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012313:RNA; D005982:Glutathione Transferase; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse