Co-expressed presenilin 1 NTF and CTF form functional gamma-secretase complexes in cells devoid of full-length protein.
The enzyme gamma-secretase catalyzes the intramembrane proteolytic cleavage that generates the amyloid beta-peptide from the beta-amyloid precursor protein. The presenilin (PS) protein is one of the four integral membrane protein components of the mature gamma-secretase complex. The PS protein is itself subjected to endoproteolytic processing, generating stable N- and C-terminal fragment (NTF and CTF, respectively) heterodimers. Here we demonstrate that coexpression of PS1 NTF and CTF functionally mimics expression of the full-length PS1 protein and restores gamma-secretase activity in PS-deficient mammalian cells. The coexpressed fragments re-associate with each other inside the cell, where they also interact with nicastrin, another gamma-secretase complex component. Analysis of gamma-secretase activity following the expression of mutant forms of NTF and CTF, under conditions bypassing endoproteolysis, indicated that the putatively catalytic Asp257 and Asp385 residues have a direct effect on gamma-secretase activity. Moreover, we demonstrate that expression of the wild-type CTF rescues endoproteolytic cleavage of C-terminally truncated PS1 molecules that are otherwise uncleaved and inactive. Recovery of cleavage is critically dependent on the integrity of Asp385. Taken together, our findings indicate that ectopically expressed NTF and CTF restore functional gamma-secretase complexes and that the presence of full-length PS1 is not a requirement for proper complex assembly.
Laudon
Hanna
H
Karolinska Institutet, Neurotec, Section for Experimental Geriatrics, Novum, Huddinge, Sweden.
Mathews
Paul M
PM
Karlström
Helena
H
Bergman
Anna
A
Farmery
Mark R
MR
Nixon
Ralph A
RA
Winblad
Bengt
B
Gandy
Samuel E
SE
Lendahl
Urban
U
Lundkvist
Johan
J
Näslund
Jan
J
D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D017930:Genes, Reporter; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D053764:Presenilin-1; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D013329:Structure-Activity Relationship
D016229:Amyloid beta-Peptides; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D053764:Presenilin-1; D011993:Recombinant Fusion Proteins; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); C413604:nicastrin protein; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509282:Bace1 protein, mouse